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.     

Von Willebrand factor propeptide makes it easy to identify the shorter Von Willebrand factor survival in patients with type 1 and type Vicenza von Willebrand disease

Reduced von Willebrand factor (VWF) half-life has been suggested as a new pathogenic mechanism in von Willebrand disease (VWD). The usefulness of VWF propeptide (VWFpp) in exploring VWF half-life was assessed in 22 type 1 and 14 type Vicenza VWD patients, and in 30 normal subjects, by comparing the findings on post-Desmopressin (DDAVP) VWF t(1/2) elimination (t(1/2el)). The VWFpp/VWF antigen ratio (VWFpp ratio) was dramatically increased in type Vicenza VWD (13.02 +/- 0.49) when compared to normal subjects (1.45 +/- 0.06), whereas it appeared to be normal in all type 1 VWD patients (1.56 +/- 0.7), except for the four carrying the C1130F mutation (4.69 +/- 0.67). A very short VWF t(1/2el) was found in type Vicenza VWD (1.3 +/- 0.2 h), while all type 1 VWD patients had a t(1/2el) similar to that of the controls (11.6 +/- 1.4 and 15.4 +/- 2.5 h respectively), except for the four patients carrying the C1130F mutation, who had a significantly shorter VWF survival (4.1 +/- 0.2 h). A significant inverse correlation emerged between VWFpp ratio and VWF t(1/2el) in both VWD patients and normal subjects. The VWFpp ratio thus seemed very useful for distinguishing between type 1 VWD cases with a normal and a reduced VWF survival, as well as for identifying type Vicenza VWD.     

A new mutation,S1285F,within the A1 loop of von Willebrand factor induces a conformational change in A1 loop with abnormal binding to platelet GPIb and botrocetin causing type 2M von Willebrand disease

We report the identification of a new mutation in exon 28 of the von Willebrand factor (VWF) gene in two related patients with type 2M von Willebrand disease (VWD). The molecular abnormality changes the Ser 1285 to Phe within the A1 loop of VWF. The S1285F mutation was reproduced by site-directed mutagenesis on the full-length VWF cDNA. The mutated recombinant VWF (rVWF), F1285rVWF, and the hybrid, S/F1285rVWF, were expressed in COS-7 cells. F1285rVWF exhibited a slight decrease of high-molecular-weight multimers and markedly reduced ristocetin- or botrocetin-induced binding of VWF to platelets in association with a decreased binding to botrocetin. The hybrid S/F1285rVWF showed a normal multimeric profile and bound to platelets in a similar way to the patients' plasma VWF, in the presence of ristocetin or botrocetin. Thus, the new S1285F mutation within the A1 loop was responsible for the type 2M VWD observed in these patients, and was involved in the binding of VWF to botrocetin and to platelet glycoprotein Ib (GPIb). Three anti-VWF monoclonal antibodies, with conformational epitopes within the A1 loop but distinct GPIb binding inhibitory properties, showed a different interaction with F1285rVWF. These results indicate that the S1285F substitution alters the folding of the A1 loop and prevents the correct exposure of the VWF binding sites to botrocetin and GPIb.     

Evaluation of an automated platelet‐based assay of ristocetin cofactor activity

Summary. von Willebrand’s disease (VWD) is regarded as the most common congenital bleeding disorder, and although not available in all laboratories von Willebrand factor (VWF) activity is most frequently assessed as ristocetin cofactor (VWF:RCo). This test can be technically challenging, is subject to poor sensitivity (～20 IU dL^?1 VWF:RCo) and has a high degree of inter‐ and intra‐assay imprecision [coefficient of variation (cv) > 25%]. We studied an automated assay using a combined fixed platelet/ristocetin reagent (BC von Willebrand reagent, Siemens Healthcare Diagnostics) on the CS‐2000i analyser (Sysmex UK Ltd). Initially inter‐ and intra‐assay imprecision was assessed. The automated method showed good day‐to‐day reproducibility and linearity of standard curves. This technique, also gave low intra‐ and inter‐assay imprecision using commercial normal (cv < 4.5%) and pathological (cv < 8.1%) control plasmas. We then compared automated technique results from 30 healthy normal subjects and 39 VWD patients to those obtained using standard aggregometry (Bio/Data, PAP4) with lyophilised fixed platelets (Helena BioSciences) and ristocetin (American Biochemical and Pharmaceutical Ltd). The automated method had a sensitivity limit of approximately 10 IU dL^?1 vs. 20 IU dL^?1 for aggregometry. Samples giving results within the aggregometry measurable range (n = 50) exhibited good correlation with the automated technique (median 70 IU dL^?1, range 7–184 IU dL^?1; and 64 IU dL^?1, 6–138 IU dL^?1 respectively; R² = 0.85). We subsequently compared 3 different batches of BC von Willebrand reagent, using a second group of normal subjects and VWD patients (n = 35, 55–139 IU dL^?1 and n = 30, <10–50 IU dL^?1). The CS‐2000i results exhibited no clinically significant variation between batches (mean cv = 7%). The automated VWF:RCo assay offers a more sensitive, reproducible, robust and less laborious alternative to standard aggregometry.     

Measurement of von Willebrand factor-FVIII binding activity in patients with suspected von Willebrand disease type 2N: application of an ELISA-based assay in a reference laboratory

Summary.  Laboratory diagnosis of von Willebrand disease type 2N (VWD2N) is based on costly mutation analysis or in vitro measurement of the ability of plasma von Willebrand factor (VWF) to bind exogenous factor VIII (FVIII); however, the VWF-FVIII binding activity assay is complex and not widely used. Our aim was to assess the utility of the in-house VWF-FVIII binding assay in the investigation of patients with suspected VWD2N. A previously described ELISA-based FVIII binding method was simplified and adapted for the clinical laboratory use by optimizing incubation time, reagent concentrations and assay standardization. The assay was validated using samples from eight individuals with known homozygous or heterozygous VWD2N mutations, and 100 healthy adults. An additional 392 patient samples were tested, including 314 with FVIII activity <50% of normal and 78 received for routine VWF-FVIII binding activity testing. Intra- and inter-assay variations were less than 10% and 17%, respectively, and the limit of quantification was estimated as 0.12. The reference range for healthy adults was 0.73–1.42. VWF:FVIII binding activity was consistent with the genotype in subjects with available genetic data, being low in three individuals with homozygous mutation (<0.12) and intermediate in five heterozygous individuals (0.44–0.61). Screening of the 392 clinical samples identified reduced VWF:FVIII binding in 19 subjects. This assay provides accurate measurement of VWF:FVIII binding activity and successfully identifies homozygous VWD2N patients and heterozygous carriers. Use of this ELISA-based assay may help avoid the need for mutation analysis in patients with unexplained low FVIII activity.     

Use of ristocetin cofactor activity in the management of von Willebrand disease

von Willebrand disease (vWD), the most common of the hereditary bleeding disorders, arises from quantitative or qualitative defects in von Willebrand factor (vWF). vWF is a multimeric plasma protein that plays a key role in primary and secondary haemostasis. In the current classification scheme, vWD is divided into six subtypes that are based on the nature of the vWF defect. Therapeutic strategies depend on the accurate identification of these subtypes. In most clinical situations, desmopressin is effective treatment for the great majority of patients with mild (type 1) disease, while replacement therapy with factor VIII/vWF concentrates that contain high levels of vWF activity is required for most type 2 and nearly all type 3 vWD patients. Several factor VIII/vWF replacement products are available, one of which (Humate P) has been approved for the treatment of vWD by the US Food and Drug Administration. Preliminary results of recent studies support the hypothesis that treatment with factor VIII/vWF concentrates based upon the content of vWF activity as reflected in the ristocetin cofactor assay is practicable, safe and efficacious. The establishment of optimal treatment regimens with respect to dose intensity and duration will require further study.     

Type 2N von Willebrand disease: Characterization and diagnostic difficulties

Introduction

An abnormal factor VIII (FVIII) binding capacity of von Willebrand factor (VWF) identifies type 2N von Willebrand disease (VWD). Type 2N VWD patients are identified by means of the VWF FVIII binding (VWF:FVIIIB) assay, and especially their VWF:FVIIIB/VWF:Ag ratio (VWF:FVIIIB ratio).

Aim

We report on our 15‐year experience of diagnosing type 2N VWD.

Methods

We have performed 2178 VWF:FVIIIB assays in bleeders and normal subjects.

Results

von Willebrand factor (VWF):FVIIIB was reduced in 682, but only 60 had low VWF:FVIIIB ratios (<0.74). Among nine patients who had a VWF:FVIIIB ratio below 0.3, four had normal VWF levels and were homozygotes for the p.R854Q mutation; the other five had low VWF levels due to a quantitative VWF mutation combined with p.R854Q. The VWF:FVIIIB ratio ranged between 0.3 and 0.73 in 51 subjects; 34 of them were heterozygotes for the p.R854Q mutation, while one carried the p.R760C. The heterozygotes for type 2N included subjects with or without bleeding symptoms, the former with significantly lower mean VWF levels than the latter. Among the 116 normal subjects tested, six were heterozygotes for the p.R854Q mutation (all asymptomatic).

Conclusions

The prevalence of type 2N in our VWD cohort was 2.5%, and 5.2% of the general population in Northeast Italy was found heterozygous for the p.R854Q mutation. It might be difficult to reveal a type 2N defect using routine tests alone, especially when it is combined with a quantitative VWF mutation. Accordingly, we always recommend VWF:FVIIIB assay in the diagnostic workup of VWD.     

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.     

Effect of ABO blood group on the collagen-binding assay for von Willebrand factor

von Willebrand disease (VWD) is the most common congenital bleeding disorder and is caused by a quantitative or qualitative abnormality of von Willebrand factor (VWF). Ristocetin cofactor (RCoF) assay is used to evaluate VWF activity, but it does not assess collagen-binding activity. Normal values of RCoF and VWF antigen vary with ABO blood group type. The collagen-binding assay (CBA) measures VWF activity; however, its relationship with ABO blood group has not been completely explored. We performed CBA on plasma samples from 131 healthy volunteers to determine if CBA values correlated with blood type. Individuals with blood group O had a mean CBA value of 94 +/- 28%, which was significantly different from the mean of 117 +/- 33% in persons with non-O blood groups (P = 0.0001). Thus, CBA values appear to correlate with ABO blood type in a manner similar to RCoF.     

Von Willebrand factor testing ratios in the diagnosis and subtyping of von Willebrand disease

Von Willebrand disease (VWD) is a common bleeding disorder of platelet adhesion with six currently recognized subtypes. Laboratory diagnosis consists of an initial test panel including antigen, activity and factor VIII measurements, sometimes followed by further specialized testing. VWF activity/antigen testing ratios help to differentiate type 1 and type 2 disease, which is important for selection of proper therapy. Recommended ratio cutoffs differ by guideline, ranging from 0.5 to 0.7, with 0.7 commonly recommended. The ratio cutoff used affects the sensitivity and specificity for type 2 diagnosis. Variability in VWD due to underlying mutations and patient factors, as well as variability in VWF tests, impact the accuracy of ratios for VWD subtyping. This review discusses the use of activity/antigen ratios in the diagnosis and subtyping of VWD with a focus on technical aspects of the tests.     

Significant linkage and non-linkage of type 1 von Willebrand disease to the von Willebrand factor gene

Significant linkage of types 2A and 2B von Willebrand disease (VWD) to the von Willebrand factor (VWF) gene have been reported, as well as mutations in the VWF gene. However, data for the partial quantitative variant are less consistent. An inconsistency of association between the type 1 VWD phenotype and genotype has been reported recently. We undertook linkage analysis of 12 families with definite or possible type 1 VWD patients. One family with classic type 1 VWD had a high lod score (Z = 5.28, theta = 0.00). A total lod score of 10.68 was obtained for the four families with fully penetrant disease. In two families linkage was rejected, while three families did not show conclusive evidence of linkage. This study corroborates ABO blood group influence, especially in patients with mild deficiencies and/or incomplete penetrance. Indirect genetic analysis may be an option for diagnosing asymptomatic or presymptomatic type 1 VWD carriers, particularly in families showing higher penetrance. The study indicates defects of the VWF locus are to be expected in more than half of the families studied. However, as defects at different loci may be the cause of this phenotype, the results of the segregation analyses should be interpreted with caution, especially in studies involving small families, or mild expressions of the disorder or incomplete penetrance.     

Progress in large-scale purification of factor VIII/von Willebrand factor concentrates using ion-exchange chromatography

Background and Objectives We investigated and optimized the parameters of a chromatographic process suitable for industrial scale to obtain a highly purified factor VIII (FVIII)/von Willebrand factor (VWF) concentrate. Materials and Methods Several chromatographic runs were performed on the same production intermediate using different anion-exchange supports. The best matrix was selected and the final product was characterized. Once the chromatographic medium was chosen, the other parameters were evaluated to obtain the highest purified product and to modulate the VWF content in the FVIII/VWF complex. Results Fractogel EMD TMAE was the best support among those tested. It was the only one maintaining good results either with standard or double loading and flow rate conditions with respect to a typical industrial process. The chromatographic recovery of FVIII co-purified with VWF was at least 86% with a specific activity not lower than 140 IU/mg. The FVIII/VWF complex obtained is highly pure and, with the exception of immunoglobulin M (IgM), all investigated contaminant proteins are under the detection limit. Different concentrates characterized by variable FVIII/VWF ratios were purified by varying the chromatographic conditions. Conclusions Several highly purified products, suitable for haemophilia A and von Willebrand disease management, can be obtained, through the same chromatographic process, on an industrial scale.     

von Willebrand disease type IIC with different abnormalities of von Willebrand factor in the same sibship

A family with von Willebrand disease has been identified in which different members of the same sibship exhibit different abnormalities of von Willebrand factor (vWF). The two most severely affected sibs (bleeding time over 20 min) had abnormalities of vWF similar to those seen in type IIC. The smallest detectable multimer was increased and the triplet structure of individual multimers was replaced with a single band. The largest multimers could not be detected and there were relatively more small multimers than intermediate sized forms. vWF antigen (vWF:Ag) was decreased to 12.5-17% by electroimmunoassay (EIA) and to 3.2-5.5% by immunoradiometric assay (IRMA). In the less severely affected sibling (bleeding time 12.5 min) there was a similar relative increase in the smallest detectable multimer. However, the larger multimers were present and the relative concentration of large to small multimers was similar to normal. The triplet structure was altered in that the relative proportion of satellite bands to the central predominant band was decreased. vWF:Ag concentrations were moderately decreased (40-80% by EIA and 25-35% by IRMA). The father and grandfather showed a vWF multimeric pattern similar to the less severely affected sibling but there was no decrease in vWF:Ag concentration and their bleeding times were normal. These observations suggest that the interplay of several genetic factors is responsible for the expression of von Willebrand disease in this family.     

Biologic response to subcutaneous and intranasal therapy with desmopressin in a large Amish kindred with Type 2M von Willebrand disease

Summary.  The aim of this study was to characterize the adequacy and longevity of biological response to desmopressin (DDAVP) in a large Amish kindred of Type 2M von Willebrand disease (VWD) possessing C‐to‐T transition at nucleotide 4120 in exon 28 of A1 domain of von Willebrand factor (VWF) gene. Response to both intranasal (Stimate^®) and subcutaneous DDAVP administration was assessed. Rise in ristocetin cofactor activity (VWF:RCo) ≥ 40% at 90‐min post‐Stimate^® and 1–2 h after subcutaneous DDAVP was defined as initial response; response longevity was assessed only after subcutaneous dosing by measuring VWF:RCo levels at time‐points 1, 2, 4 and 6 h. Eleven patients (five males, six females; age range: 20–56 years) participated in intranasal and 9/11 (four males, five females) in subcutaneous testing. Baseline haemostatic profiles included: VWF:RCo < 15%, VWF:Ag < 40% and normal VWF multimers. Initial response was comparable by both intranasal (6/11; 54.5%) and subcutaneous (4/9; 44%) routes; sustained response (VWF:RCo > 40% for 2 h) was observed in only one in nine (11%) patients tested. Median VWF:RCo peak levels after intranasal (40%) and subcutaneous (39%) routes were equivalent. Peak VWF:Ag levels were significantly higher after subcutaneous than intranasal DDAVP (94% vs. 54%; P = 0.03). Area under the curve for VWF:RCo was significantly decreased (170 μg h mL^?1) compared with VWF:Ag (471 μg h mL^?1) and FVIII:C (624.60 μg h mL^?1). This study suggests that in this population: (i) intra‐individual DDAVP response is consistent with subcutaneous and intranasal administration; and (ii) extending DDAVP challenge test up to at least 6 h is required to characterize adequacy and longevity of biologic response prior to using DDAVP as a sole haemostatic intervention.     

Screening for von Willebrand disease: contribution of an automated assay for von Willebrand factor activity

Measuring von Willebrand factor (VWF) activity is essential to the diagnosis of von Willebrand disease (VWD). The VWF activity is usually assessed based on measurement of the ristocetin cofactor (VWF:RCo). However, that test is technically challenging and has high intra- and inter-assay variabilities. The HemosIL VWF activity (VWF:AC) is a fully automated assay, recently proposed as a good alternative to VWF:RCo for VWD diagnosis. This study was undertaken to assess this new method. First, the analytical performance of VWF:AC on an automated coagulo-meter (ACLTop) was determined, and then this new method was compared with VWF:RCo and the platelet function analyzer (PFA100) for 160 patients referred for VWD screening. The VWF:AC achieved acceptable precision with within-run and between-run coefficients of variation ranging from 2.3% to 14.1%, and linearity from 10% to 100%. Despite some marked differences between VWF:AC and VWF:RCo for 10 plasmas tested, their agreement for VWD diagnosis was good. The VWF:AC had sensitivity similar to that of PFA100 (close to 100%), but better specificity (97.7% vs. 66% or 60%, depending on the cartridge used). The good analytical performance, and the sensitivity and specificity of VWF:AC to detect VWF deficiency renders it a suitable method for VWD screening. Our findings support VWF:AC use for the diagnostic work-up of VWD, paying close attention to concomitant clinical signs and bleeding score, as recommended for 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.