Changes in von Willebrand factor level and von Willebrand activity with age in type 1 von Willebrand disease

In a normal population, VWF plasma levels (VWF:Ag) and VWF activity (VWF:RCo) increase by approximately 0.17 and 0.15 IU mL^?1 per decade, but the influence of age is unknown in patients with type 1 von Willebrand disease (VWD). In a retrospective cohort study, the medical records of 31 type 1 VWD patients over the age of 30, who had been followed for ≥5 years, were reviewed for baseline clinical data and previously performed VWF:Ag, VWF:RCo and factor VIII levels (FVIII:C). VWF multimer analysis was normal in 28/31 cases performed. Mean age at diagnosis was 33 (range 16–60 years), and duration of follow‐up ranged from 5 to 26 years (mean 11 years). Patients had 2–10 time points of VWD testing (mean of 5.2). The mean VWF:Ag, VWF:RCo and FVIII:C at time of diagnosis were 0.44 IU mL^?1 0.34 IU mL^?1 and 0.75 IU mL^?1. At last follow‐up, the mean VWF:Ag, VWF:RCo and FVIII:C were significantly increased to 0.71 IU L^?1, 0.56 IU mL^?1 and 0.90 IU mL^?1 (P ≤ 0.001, <0.001, and 0.0081 respectively). Here 18/31 patients had VWF:Ag, VWF:RCo and FVIII: C levels that increased into the normal range. The rate of change in VWF:Ag, VWF:RCo and FVIII was 0.30 IU mL^?1 (0.21–0.39, CI 95%, P < 0.0001), 0.20 IU mL^?1 per decade (0.13–0.27, CI 95%, P = 0.0001) and 0.20 IU mL^?1 (0.11–0.29, CI 95%, P = 0.0011). Patients with type 1 VWD experience age‐related increases to VWF:Ag and VWF:RCo which can result in normalization of VWF levels. Further studies are required to determine if the bleeding phenotype resolves with the increases in VWF:Ag and VWF:RCo levels.     

Outgrowing the laboratory diagnosis of type 1 von Willebrand disease: A two decade study

Von Willebrand Factor (VWF) levels are known to increase with age in the general population, but that effect is unclear in von Willebrand disease (VWD) patients. Thus, it is important to assess the trends of VWF levels with age, and the extent and rate of their normalization in patients with VWD. In a retrospective cohort study, we reviewed the medical records of 126 patients between 1996 and 2016 who met the NHLBI diagnostic criteria for type 1 VWD or “Low VWF” (LVWF). We followed all their historically documented VWF antigen (VWF:Ag), VWF activity (VWF:RCo), and Factor VIII (FVIII) levels longitudinally over time, correlating data with clinical setting at time of testing. The average duration of follow‐up was 10.5 ± 3.7 years (SD). Out of the total study population, 27.8% achieved the primary outcome of complete normalization (CN) of both VWF:Ag and VWF:RCo levels, including 19.6% and 32.5% of those with VWD and LVWF, respectively. Linear regression demonstrated statistically significant positive trends of VWF:Ag, VWF:RCo, FVIII with time, calculated at 2.4, 1.4, and 1.4 U dL‐1/year, respectively (P < .001 each). In the largest study population of VWD patients to date whose levels were followed longitudinally, there is a statistically significant rise in VWF:Ag, VWF:RCo, and FVIII levels observed with time. CN of both VWF:Ag and VWF:RCo levels was observed in almost a third of patients with VWD or LVWF, over an average of 10 years. Whether the bleeding phenotype also improves is unclear and requires further study.     

von Willebrand factor/factor VIII concentrate (Humate‐P) for management of elective surgery in adults and children with von Willebrand disease

Summary. von Willebrand disease (VWD) is the most common inherited bleeding disorder. Treatment guidelines recommend the use of von Willebrand factor/factor VIII (VWF/FVIII) concentrate for VWD patients with type 2 or 3 VWD undergoing surgery, and type 1 patients undergoing surgery who are unresponsive, or for whom desmopressin acetate is contraindicated. This prospective, open‐label, multinational study evaluated the safety, efficacy and optimal dosing of a VWF/FVIII concentrate (Humate‐P) in subjects with VWD undergoing elective surgery. Dosing was based on VWF ristocetin cofactor (VWF:RCo) and FVIII pharmacokinetic assessments performed before surgery. Pharmacokinetic assessments were completed in 33 adults and 9 children. Haemostatic efficacy was assessed on a 4‐point scale (excellent, good, moderate/poor or none). Overall effective haemostasis was achieved in 32/35 subjects. Median terminal VWF:RCo half‐life was 11.7 h, and median incremental in vivo recovery was 2.4 IU dL^?1 per IU kg^?1 infused. Major haemorrhage occurred after surgery in 3/35 cases despite achieving target VWF and FVIII levels. Median VWF/FVIII concentrate loading doses ranged from 42.6 IU VWF:RCo kg^?1 (oral surgery) to 61.2 IU VWF:RCo kg^?1 (major surgery), with a median of 10 (range, 2–55) doses administered per subject. Adverse events considered possibly treatment‐related (n = 6) were generally mild and of short duration. The results indicate that this VWF/FVIII concentrate is safe and effective in the prevention of excessive bleeding during and after surgery in individuals with VWD.     

A comparative in vitro evaluation of six von Willebrand factor concentrates

The efficacy of von Willebrand factor (VWF) concentrates for treatment of von Willebrand disease (VWD) is dependent on their content of VWF and factor VIII (FVIII). STUDY OBJECTIVES: To measure the content and quality of VWF and FVIII in six VWF concentrates: Haemate-P (Aventis Behring), Immunate (Baxter Bioscience), Koate (Bayer Corp.), 8Y (BPL), Innobrand (LFB) and Facteur Willebrand (LFB). METHODS: The VWF antigen content (VWF:Ag), ristocetin cofactor activity (VWF:RCo), collagen-binding activity (VWF:CB), VWF multimers with electrophoresis and densitometry, FVIII activity and total protein content. RESULTS: Specific activity (VWF:RCo/total protein) varied considerably (4.7-129.5 IU mg(-1)). Activity measures, VWF:RCo and VWF:CB, correlated well, but we found no correlation between any of these and VWF:Ag. The content of high-molecular weight multimer (HMWM) was normal or close to normal in Haemate-P, Innobrand and Facteur Willebrand, moderately reduced in Koate and 8Y, and significantly reduced in Immunate. The HMWM content correlated significantly with the VWF:RCo/VWF:Ag ratio. Only Haemate-P, Innobrand and Facteur Willebrand had VWF:RCo/VWF:Ag ratios >0.7. We found large differences in the content of FVIII and in the FVIII/VWF:RCo ratio. Facteur Willebrand had the lowest (0.02) and Immunate the highest (6.00) ratio. CONCLUSION: Treating physicians must be aware of the large differences between different VWF concentrates and the potential clinical implications. Concentrates lacking HMWM are probably less efficient for mucosal bleedings. FVIII is most important for surgical bleedings, but concentrates with high FVIII/VWF-ratio may induce very high FVIII levels with increased risk of thrombosis. A low FVIII content may be preferable except in case of acute surgery.     

A two‐centre comparative evaluation of new automated assays for von Willebrand factor ristocetin cofactor activity and antigen

von Willebrand disease (VWD) is caused by a quantitative and/or qualitative deficiency of the von Willebrand factor (VWF). The laboratory diagnosis of VWD is dependent on the measurement of VWF antigen (VWF:Ag) and ristocetin cofactor activity (VWF:RCo). The aim of this study was to undertake a two‐centre evaluation of two new automated VWF:Ag and VWF:RCo assays systems from Instrumentation Laboratory (Bedford, USA). Using the two new analytical systems that operated with different detection principles: immunoturbidimetric (TOP500 analyser) and chemiluminescent (AcuStar analyser), VWF:Ag and VWF:RCo levels were determined in samples from 171 healthy normal subjects, 80 VWD patients (16 type 1, 58 type 2 and 6 type 3) and 7 acquired von Willebrand syndrome patients. With commercial lyophilized normal and pathological plasmas VWF: Ag and VWF:RCo assays performed on both analysers exhibited low levels of inter‐assay imprecision (AcuStar: CV% range 3.3–6.9; TOP500: CV% range 2.6–6.3). Samples from normal healthy subjects (range: VWF:Ag 44.6–173.9 IU dL^?1; VWF:RCo 43.1–191.5 IU dL^?1) and patients (range: VWF:Ag <0.3–115.1 IU dL^?1; VWF:RCo <0.5–57.2 IU dL^?1) showed a good correlation between the two VWF:Ag and VWF:RCo methods (r_s = 0.92 and 0.82 respectively), with only a few inconsistent cases among the patients' samples evaluated. The chemiluminescent assays had a lower limit of detection for both VWF:Ag and VWF:RCo compared to immunoturbidimetric tests (0.3 IU dL^?1 vs. 2.2 IU dL^?1 and 0.5 IU dL^?1 vs. 4.4 IU dL^?1 respectively). The TOP500 and AcuStar VWF:Ag and VWF:RCo assays were precise and compare well between centres, making these systems suitable for the diagnosis of VWD in non‐specialized and reference laboratories.     

Laboratory diagnostic approach of the parents-children relationship in differentiating low-level von Willebrand factor from mild type 1 von Willebrand disease

The present study aimed to evaluate the parent-child relationship in differentiating between unaffected healthy individuals and those with von Willebrand disease (VWD). This study was performed on 15 children between the ages of 5 and 15 years and parents with personal and familial evidence of bleeding. Diagnosis of VWD as considered 'low von Willebrand factor (VWF) level or mild type 1 VWD' in the following children: those with low VWF levels (VWF:RCo and VWF:Ag between 30 and 50 U/dl), at least one bleeding symptom and a family member with at least one bleeding symptom. Laboratory values in the parents of families 1-7 were VWF:Ag 65-90, VWF:RCo 54-87, and FVIII:C 74-110, versus VWF:Ag 33-47, VWF:RCo 30-42, and FVIII:C 36-67 in their children. The normal laboratory values in the parents of families 1-7 suggested that their children would probably have low VWF levels. Our findings are that VWF levels are increasing with age. Laboratory values in the parents of families 8-15 were VWF:Ag 30-59, VWF:RCo 32-55, and FVIII:C 44-66, versus VWF:Ag 32-48, VWF:RCo 30-54, and FVIII:C 38-55 in their children. The laboratory values in the children from families 8-15 were close to the minimum range of normal or below normal, which suggested that it was possible that the parents and children in families 8-15 could be diagnosed as having mild type 1 VWD. The present study's findings show that comparison of the VWF levels in parents and their children may be helpful in differentiating children with low VWF levels and mild type 1 VWD from children that only have low VWF levels.     

Presurgical pharmacokinetic analysis of a von Willebrand factor/factor VIII (VWF/FVIII) concentrate in patients with von Willebrand’s disease (VWD) has limited value in dosing for surgery

Summary. Optimal doses of von Willebrand Factor/Factor VIII (VWF/FVIII) concentrates for surgical procedures in patients with VWD need to be determined. A prospective, multicenter study was performed that included an initial pharmacokinetic (PK) assessment following a standard dose of VWF/FVIII concentrate (Humate‐P^®) to determine individual PK parameters and guide therapeutic dosing during surgery. Forty one subjects received 60 IU kg^?1 VWF: RCo. Median plasma levels, half‐life, mean change from baseline and in vivo recovery (IVR) values were determined for VWF:RCo, VWF:Ag, and FVIII: C, and area under the plasma time‐concentration curve (AUC), mean residence time (MRT), clearance, volume of distribution and dose linearity were also assessed for VWF:RCo at various time points. Median baseline VWF:RCo level was 13 IU dL^?1 (range, 6–124); with a mean change from baseline >100 IU dL^?1 immediately after the infusion, decreasing to 10 IU dL^?1 at 48 h postinfusion. The group median incremental in vivo recovery (IVR) for VWF:RCo was 2.4 IU dL^?1 per IU kg^?1, for VWF:Ag 2.3 IU dL^?1 kg^?1 and for FVIII:C was 2.7 IU dL^?1 per IU kg^?1. When analysing individual recovery values on repeated infusions, a very weak correlation was observed between presurgery IVR and IVR for both VWF:RCo and FVIII, measured at various times just prior to and after the surgical procedure. Although group median values were fairly consistent among repeated IVR measurements, the intra‐individual IVR values for FVIII and VWF:RCo with repeated infusions showed a large degree of variability. IVR values obtained from pharmacokinetic analyses performed in advance of anticipated surgery do not reliably predict postinfusion circulating levels of VWF:RCo or FVIII attained preoperatively or with subsequent peri‐operative infusions.     

Diagnosis and management of adult patients with von Willebrand disease in South Australia

We have analyzed the databases for von Willebrand disease (VWD) from the hemophilia center for adult patients with bleeding disorders in South Australia. We define the prevalence of types of VWD to determine the proportion of who would be treated with factor (F) VIII/von Willebrand factor (VWF) concentrate to prevent or control hemorrhage. In severe or moderately severe patients, we use plasma-derived FVIII/VWF concentrate, and for mild to severe cases, we use desmopressin plus tranexamic acid. There are 103 patients with VWF ristocetin (RCo) ≤50 IU/dL: 38 (37%) severe (VWF:RCo <10 IU/dL), 28 (27%) moderate (VWF:RCo 10 to 29 IU/dL), and 37 (36%) mild (VWF:RCo 30 to 50 IU/dL). Hence in 66 (64%), FVIII/VWF concentrate is the mainstay of treatment. The prevalence of VWD in our region according to data from our center is ~1 per 12,000. A total of 52% of patients are type 1, 44% type 2, and 5% type 3. In our experience, type 2M (45% of type 2) is much more common than types 2A and 2B (each 9% of type 2). Mutation detection is useful for identifying some subtypes of VWD.     

Postpartum von Willebrand factor levels in women with and without von Willebrand disease and implications for prophylaxis

The aim of this study was to elucidate the fall in von Willebrand factor (VWF) and factor VIII activity (FVIII) after childbirth in women with and without von Willebrand disease (VWD). VWF:RCo, VWF:Ag, and FVIII were obtained in the third trimester of pregnancy, on admission for childbirth, and 10 times postpartum. Specimens were processed within 4 h and analysed centrally. Means were calculated at each time point. Forty women (40 pregnancies) without VWD and 32 women (35 pregnancies) with VWD were enrolled. 15/32 with VWD were treated (30% of those with type 1 and all of those with type 2) in 17 pregnancies. Treatments prior to delivery consisted of desmopressin (2/17), VWF concentrate (15/17) and after delivery VWF concentrate (16/17). Duration of treatment was 0–21 days (median 6). VWF levels peaked at 250% of baseline – 4 h postpartum in women with VWD and 12 h postpartum in women without VWD. Thereafter, VWF levels fell rapidly, approached baseline at 1 week and reached baseline at 3 weeks. Except immediately postpartum, when the levels among treated cases were higher, levels among women with VWD appeared to parallel, but were lower than those among women without VWD. Levels were lowest among those who received treatment. VWF levels fall rapidly after childbirth. Except immediately postpartum, current treatment strategies do not raise VWF levels to the levels of women without VWD or even to the levels of women with milder, untreated VWD. Consequently, women with VWD may be at risk of postpartum haemorrhage despite treatment.     

A comparative evaluation of a new fully automated assay for von Willebrand factor collagen binding activity to an established method

Introduction

Laboratory diagnosis of von Willebrand disease (VWD) is made by the measurement of von Willebrand factor (VWF) protein level and its activities. Current VWF activity tests include ristocetin cofactor and collagen binding (VWF:CB) assays.

Aim

We have undertaken an evaluation of a new fully automated VWF:CB assay relative to an established enzyme‐linked immunosorbent assay (ELISA) method.

Methods

The two analytical systems operate with different detection principles: a chemiluminescent method performed on ACL AcuStar Analyzer (the former) and a colorimetric ELISA by Asserachrom Stago (the latter) (type III collagen from human placenta). The HemosIL AcuStar VWF:CB assay is a chemiluminescent 2‐step immunoassay that uses magnetic particles coated with a type III collagen triple‐helical peptide. VWF:CB levels were determined in 50 healthy subjects and 100 VWD patients (22 type 1, 73 type 2 and 5 type 3).

Results

Eleven VWD samples reported VWF:CB values below the lower detection limit of one or both methods. The new method showed a good correlation with the ELISA method (r > .9, mean bias 3.85 IU/dL) in both healthy and VWD samples. One of 150 samples gave inconsistent results using the two assays, leading to an uncertain diagnosis of VWD type 1 (ELISA method) or type 2 MCB (fully automated method).

Conclusion

The new assay is rapid and simple to use, with its ready‐to‐use reagent cartridges. This VWF:CB assay, in addition to the measurement of VWF:Ag and VWF:RCo made on the same platform, gives additional information for the diagnosis of VWD in both nonspecialized and reference laboratories.     

Diagnosis of inherited von Willebrand disease: comparison of two methodologies and analysis of the discrepancies

Diagnostics of von Willebrand disease (VWD) includes assessment of factor VIII (FVIII) coagulant activity, von Willebrand factor (VWF) antigen (VWF:Ag) and VWF ristocetin cofactor activity (VWF:RCo), and more specific tests as multimeric and genetic analyses are necessary for the correct VWD classification. The ACL AcuStar? analyzer introduces chemiluminescence (CL) technology in detection of VWD with automated VWF:Ag and VWF:RCo assays. Compare VWF:Ag‐ELISA and VWF:RCo by aggregometry conventional assays with new CL VWF:Ag‐IL and VWF:RCo‐IL assays, investigate the ability to make accurate VWD diagnosis and concordance with multimeric and genetic analyses. 146 patients with congenital VWD (51 Type 1; 34 Type2A; 16 Type 2B; 31 Type 2M; 5 Type 2N; 9 Type 3) and 30 healthy normal subjects were included. A comparison was made between CL and conventional methods. Diagnostic evaluation included: VWF:RCo/VWF:Ag ratio, multimeric distribution (sodium dodecyl sulfate [SDS]‐agarose gel) of VWF and genetic analysis in 110 of 146 patients. CL and conventional methods revealed good correlation. Kappa test agreement diagnosis was >0.8. CL diagnostic sensitivity was 100% and specificity 97%. Multimeric and genetic analysis were of help in clarifying 13 discrepancies of diagnosis between methods, of which six discrepancies were explained by lack of conventional methods′ sensibility. CL methodology can detect VWD and discriminate between type 1, 3 and variant forms and offers an automated, faster, sensitive and less cumbersome method when compared to conventional assays, in particular VWF:RCo by aggregometry. In some cases, even with all phenotype and genetic analyses, discrepancies exist in the classification of VWD.     

Use of the collagen-binding assay for von Willebrand factor in the analysis of type 2M von Willebrand disease: a comparison with the ristocetin cofactor assay

This study compares the utility of two functional assays for von Willebrand factor (VWF), the ristocetin cofactor assay (VWF:RCo) and the collagen-binding assay (VWF:CBA). We analysed a group of 32 patients with type 2 von Willebrand disease (VWD) (25 patients with type 2M, six with type 2A and one with type 2B) and 22 normal control subjects. VWF:RCo/VWF antigen (VWF:Ag) ratios and VWF:CBA/VWF:Ag ratios were compared between the patient and control groups. In the six patients with type 2A VWD, both VWF:RCo/VWF:Ag ratios and VWF:CBA/VWF:Ag ratios were discordant (< or = 0.7). In the 25 type 2M VWD patients, the VWF:CBA/VWF:Ag ratios were concordant (> 0.7), but the VWF:RCo/VWF:CBA ratios were discordant (< or = 0.7) (P = 0.001) compared with control subjects. Thus, VWF:RCo/VWF:Ag ratios were discordant in both type 2M and 2A VWD patient groups indicating a functional abnormality. However, VWF:CBA/VWF:Ag ratios were discordant in the type 2A VWD group but not in the type 2M VWD group. Our study showed that VWF:CBA is sensitive to functional variants associated with the loss of high-molecular-weight multimers, i.e. type 2A and 2B in VWD, but the assay was unable to discriminate defective platelet-binding VWD variants with normal multimeric patterns such as type 2M VWD. It was concluded that the VWF:CBA assay should be used in association with rather than as a replacement for the VWF:RCo assay.     

Comorbidities associated with higher von Willebrand factor (VWF) levels may explain the age‐related increase of VWF in von Willebrand disease

Some comorbidities, such as hypertension, are associated with higher von Willebrand factor (VWF) levels in the general population. No studies have been conducted to assess this association in patients with von Willebrand disease (VWD). Therefore, we studied this association in patients with type 1 (n = 333) and type 2 (n = 203) VWD from the ‘WiN” study. VWF antigen (VWF:Ag) was higher in type 1 VWD patients with hypertension [difference: 0·23 iu/ml, 95% confidence interval (CI): 0·11–0·35], diabetes mellitus (0·11 iu/ml, 95% CI: −0·02 to 0·23), cancer (0·14 iu/ml, 95% CI: 0·03–0·25) and thyroid dysfunction (0·14 iu/ml, 95% CI: 0·03–0·26) than in patients without these comorbidities (all corrected for age, sex and blood group). Similar results were observed for VWF collagen binding capacity (VWF:CB), VWF activity as measured by the VWF monoclonal antibody assay (VWF:Ab) and factor VIII (FVIII) coagulant activity (FVIII:C). In type 1 VWD, age was associated with higher VWF:Ag (0·03 iu/ml; 95% CI: 0·01–0·04), VWF:CB (0·02 iu/ml; 95% CI: 0·00–0·04), VWF:Ab (0·04 iu/ml; 95% CI: 0·02–0·06) and FVIII:C (0·03 iu/ml; 95% CI: 0·01–0·06) per decade increase. After adjustment for relevant comorbidities, these associations were no longer significant. Despite the higher VWF and FVIII levels, type 1 VWD patients with comorbidities had more bleeding episodes, particularly during surgery. There was no association between comorbidities and VWF/FVIII levels or bleeding phenotype in type 2 VWD patients. In conclusion, comorbidities are associated with higher VWF and FVIII levels in type 1 VWD and may explain the age‐related increase of VWF and FVIII levels.     

Laboratory misdiagnosis of von Willebrand disease in post-menarchal females: A multi-center study

Increased awareness of von Willebrand Disease (VWD) has led to more frequent diagnostic laboratory testing, which insurers often dictate be performed at a facility with off-site laboratory processing, instead of a coagulation facility with onsite processing. Off-site processing is more prone to preanalytical variables causing falsely low levels of von Willebrand Factor (VWF) due to the additional transport required. Our aim was to determine the percentage of discordance between off-site and onsite specimen processing for VWD in this multicenter, retrospective study. We enrolled females aged 12 to 50 years who had off-site specimen processing for VWF assays, and repeat testing performed at a consulting institution with onsite coagulation phlebotomy and processing. A total of 263 females from 17 institutions were included in the analysis. There were 251 subjects with both off-site and onsite VWF antigen (VWF:Ag) processing with 96 (38%) being low off-site and 56 (22%) low onsite; 223 subjects had VWF ristocetin co-factor (VWF:RCo), 122 (55%) were low off-site and 71 (32%) were low onsite. Similarly, 229 subjects had a Factor VIII (FVIII) assay, and 67 (29%) were low off-site with less than half, 29 (13%) confirmed low with onsite processing. Higher proportions of patients demonstrated low VWF:Ag, VWF:RCo, and/or FVIII with off-site processing compared to onsite (McNemarʼs test P-value <.0005, for all assays). These results emphasize the need to decrease delays from sample procurement to processing for VWF assays. The VWF assays should ideally be collected and processed at the same site under the guidance of a hematologist.     

Development of an ELISA method for testing VWF ristocetin cofactor activity with improved sensitivity and reliability in the diagnosis of von Willebrand disease

Objectives: Current methods in assessing von Willebrand factor (VWF) ristocetin cofactor activity for Von Willebrand’s disease (VWD) diagnosis include platelet agglutination by aggregometer or macroscopic slide examination, which are both time‐consuming with suboptimal interassay and intra‐assay variation. The purpose of this study is to establish a sensitive assay to detect VWF:RCo activity and evaluate its performance in VWD diagnosis. Methods: We have established a sensitive VWF:RCo–ELISA method using a monoclonal antibody, SZ‐151, to immobilize the recombinant fragment of platelet glycoprotein Ib (rfGPIbα). VWF was captured by rfGPIbα in the presence of ristocetin, and then detected by HRP‐conjugated rabbit anti‐human VWF IgG. We tested the VWF:RCo level by this VWF:RCo–ELISA in 25 patients with different types of VWD and 36 healthy donors, and compared this method to a previously reported ELISA using 2D4 coating antibody. Results: The sensitivity of VWF:RCo–ELISA was greatly improved with this assay (0.008 IU/dL compared to 0.031 IU/dL by 2D4 antibody). The interassay and intra‐assay coefficient variation were 8% and 12%, respectively. The mean values (ranges) of VWF:RCo in patients with type 1, type 2A, type 2B, type 2M, and type 3 of VWD and control group are 31.8 (22.3–56.9), 4.8 (0.6–11.8), 8.6 (1.6–19.7), 3.9 (1.0–6.8), 1.0 (0.5–1.6), and 91.5 (47.3–169.2) IU/dL, respectively. The corresponding ratios (ranges) of VWF:RCo / VWF:Ag are 0.83 (0.70–1.16), 0.27 (0.08–0.58), 0.31 (0.15–0.40), 0.18 (0.14–0.21), 0.52 (0.13–1.19), and 0.92 (0.62–1.26). Conclusion: The VWF:RCo–ELISA using monoclonal anti‐rfGPIbα antibody SZ‐151 showed improved sensitivity and reliability in detecting VWF:RCo activity, and its clinical application would facilitate the diagnosis and classification of VWD.     

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.     

Laboratory diagnosis and management of von Willebrand disease in Turkey: Izmir experience

Von Willebrand disease (VWD) is caused by a deficiency or dysfunction of Von Willebrand factor (VWF). The pathophysiology, classification, diagnosis, and management of VWD are relatively complex, but their understanding is important for proper diagnosis and management of patients with VWD. There are inherent difficulties in both the identification and classification of VWD because of clinical uncertainty and the limitations in the test processes and test panels typically used by laboratories. The most common test panel employed by laboratories, particularly in the geographic regions covered by the mutational studies, would comprise factor VIII coagulant (FVIII:C), VWF protein (antigen; VWF:Ag), and ristocetin cofactor (VWF:RCo). In our center, use of a desmopressin challenge with our core four-test panel (i.e., VWF:Ag, VWF:RCo, FVIII:C, and PFA-100) is expected to further assist laboratory diagnosis of VWD in Turkey. Molecular genetics is a rather new approach for Turkey, with gene analyses related to VWD being initiated in one center and the results used for confirmation of diagnosis in limited cases.     

Response to desmopressin is influenced by the genotype and phenotype in type 1 von Willebrand disease (VWD): results from the European Study MCMDM-1VWD

We have prospectively evaluated the biologic response to desmopressin in 77 patients with type 1 von Willebrand disease (VWD) enrolled within the Molecular and Clinical Markers for the Diagnosis and Management of type 1 VWD project. Complete response to desmopressin was defined as an increase of both ristocetin cofactor activity (VWF:RCo) and factor VIII coagulant activity (FVIII:C) to 50 IU/dL or higher and partial response as VWF:RCo or FVIII:C lower than 50 IU/dL after infusion, but at least 3-fold the basal level. Complete response was observed in 83% of patients; partial in 13%; and no response in 4%. Patients with some abnormality of VWF multimeric pattern had significantly lower basal FVIII:C and VWF, lower VWF:RCo/Ag ratio, and less complete responses to desmopressin than patients with a normal multimeric pattern (P=.002). Patients with mutations at codons 1130 and 1205 in the D'-D3 domain had the greatest relative increase, but shortest FVIII and VWF half-lives after infusion. Most partial and nonresponsive patients had mutations in the A1-A3 domains. Response to desmopressin in these VWD patients seemed to be associated with the location of the causative mutation. The presence of subtle multimeric abnormalities did not hamper potential clinically useful responses, as in typical type 1 VWD.     

Intravenous DDAVP and factor VIII-von Willebrand factor concentrate for the treatment and prophylaxis of bleedings in patients With von Willebrand disease type 1, 2 and 3.

The current standard set of von Willebrand factor (VWF) parameters used to differentiate type 1 from type 2 VWD include bleeding times (BTs), factor VIII coagulant activity (FVIII:C), VWF antigen (VWF:Ag), VWF ristocetine cofactor activity (VWF:RCo), VWF collagen binding activity (VWF:CB), ristocetine induced platelet aggregation (RIPA), and analysis of VWF multimers in low and high resolution agarose gels and the response to DDAVP. The BTs and RIPA are normal in asymptomatic carriers of a mutant VWF allele, in dominant type 1, and in recessive type 2N VWD, and this category has a normal response of VWF parameters to DDAVP. The response of FVIII:C is compromised in type 2N VWD. The BTs and RIPA are usually normal in type Vicenza and mild type 2A VWD, and these two VWD variants show a transiently good response of BT and VWF parameters followed by short in vivo half life times of VWF parameters. The BTS are strongly prolonged and RIPA typically absent in recessive severe type 1 and 3 VWD, in dominant type 2A and in recessive type 2C (very likely also 2D) VWD and consequently associated with low or absent platelet VWF, and no or poor response of VWF parameters to DDAVP. The BTs are prolonged and RIPA increased in dominant type 2B VWD, that is featured by normal platelet VWF and a poor response of BT and functional VWF to DDAVP. The BTs are prolonged and RIPA decreased in dominant type 2A and 2U, that all have low VWF platelet, very low VWF:RCo values as compared to VWF:Ag, and a poor response of functional VWF to DDAVP. VWD type 2M is featured by the presence of all VWF multimers in a low resolution agarose gel, normal or slightly prolonged BT, decreased RIPA, a poor response of VWF:RCo and a good response of FVIII and VWF:CB to DDAVP and therefore clearly in between dominant type 1 and 2U. The existing recommendations for prophylaxis and treatment of bleedings in type 2 VWD patients with FVIII/VWF concentrates are mainly derived from pharmocokinetic studies in type 3 VWD patients. FVIII/VWF concentrates should be characterised by labelling with FVIII:C, VWF:RCo, VWF:CB and VWF multimeric pattern to determine their safety and efficacy in prospective management studies. As the bleeding tendency is moderate in type 2 and severe in type 3 VWD and the FVIII:C levels are near normal in type 2 and very low in type 3 VWD patients. Proper recommendations of FVIII/VWF concentrates using VWF:RCo unit dosing for the prophylaxis and treatment of bleeding episodes are proposed and has to be stratified for the severity of bleeding, the type of surgery either minor or major and for type 2 and type 3 VWD as well.     

Clinical phenotype in genetically confirmed von Willebrand disease type 2N patients reflects a haemophilia A phenotype

Introduction : Von Willebrand disease (VWD) type 2N is characterized by a defective binding of factor VIII (FVIII) to von Willebrand factor (VWF) resulting in diminished plasma FVIII levels and a clinical phenotype mimicking mild haemophilia A. Several mutations in the FVIII binding site of VWF have been reported. Aim: This study aims to examine the effect of genotype on clinical phenotype in a cohort of VWD 2N patients. Methods: Patients with at least one genetically confirmed 2N mutation were selected retrospectively from a cohort of patients with suspected VWD. Clinical and laboratory phenotypes including bleeding scores (BS) were obtained and analysed. Results : Forty‐two VWD 2N patients with a mean age of 44 years were included. Eleven patients were homozygous or compound heterozygous (genetically confirmed group) and 31 patients were heterozygously affected (carriers group). Statistically significant differences between genetically confirmed VWD 2N patients and carriers were found in FVIII activity, VWF antigen levels, VWF‐FVIII binding capacity, FVIII/VWF antigen ratio (all P<0.001), VWF‐ristocetin activity (p=0.001) and VWF collagen binding (P = 0.002). Median BS was 6 in genetically confirmed VWD 2N patients compared with 3 in carriers (P = 0.047). Haemarthrosis, muscle haematomas and postpartum haemorrhage were only reported in genetically confirmed 2N patients. Conclusion : Phenotypic analysis showed that all laboratory parameters are lower in genetically confirmed VWD 2N patients compared with heterozygous 2N carriers. The clinical phenotype in genetically confirmed VWD 2N patients is comparable to mild haemophilia A patients and more severe than heterozygous 2N carriers.