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Is it time for Ristocetin to step down? Comparison study between a new automated von Willebrand factor activity assay and the von Willebrand factor ristocetin activity assay.

INTRODUCTION

von Willebrand disease (VWD) is the most common inherited haemorrhage disorder (1). The disease prevalence is not precisely known, however, it has been estimated to range from 0.01% to 1.0% in the general population (2). Furthermore, the prevalence estimation for VWD varies among haemostasis centres (3). VWD is a heterogeneous bleeding disorder that arises from either a quantitative or qualitative defect and/or a deficiency in von Willebrand factor (VWF). Like any other congenital disorders, mutations that occur in VWF gene include deletions, frameshift, splice-site, and nonsense. VWD is inherited as an autosomal disorder. The majority of type 1 and type 2A cases are inherited in a dominant fashion. However, few studies suggest recessive rather than dominant inheritance (4,5). Type 2B and 2M are autosomal dominant, while type 3 and type 2N are inherited as recessive disorders. Nevertheless, de novo mutation has been reported (6).

VWF is a large, complex, multimer plasma glycoprotein composed of repeated units that are polymerised from dimers by disulphide bonds (Figure 1) (7). VWF is manufactured in two cell types, endothelial cells and megakaryocytes (1). In endothelial cells, after being synthesised, VWF multimers are packaged into rod-shaped secretory vesicles called WeibelPalade bodies, from which it is released upon stress or following drug exposure (5). The secreted VWF from endothelial cells represents approximately 85% of the circulating VWF level (9). The 15% remainder of VWF is formed by megakaryocytes and stored in a-granules of platelets and secreted upon platelet activation (8).

VWF has a half-life of approximately 12 hours, ranging from 9 15 hours (9), and is cleared from the circulation by macrophages in the spleen and liver in a process that is independent of its size (8). Upon secretion, the ultra-large VWF multimers have a high affinity to bind to platelets spontaneously without shear stress or any other stimulants (10). Subsequently, these large multimers undergo proteolytic degradation by metalloprotease ADAMST-13 (a disintegrin and metalloprotease with thrombospondin type 1 motifs), which cleaves the molecule in domain A2 to produce circulating VWF of various multimer sizes (10,11).

Several binding sites have been identified in the VWF subunit (Figure 1). Platelet GPIb interacts with domain A1 and integrin GPII b/111 a interacts with an Arg-Gly-Asp sequence in domain C1. Fibrillar collagens interact mainly with domain A3 and collagen VI binds to domain A1. Coagulation factor VIII (FVIII) binds to the N-terminal of D'D3 region (12).

The VWF gene is located on the short arm of chromosome 12, at position 12p13.3 and spans approximately 178 kb and contains 52 exons. (13,14). Moreover, there is an unprocessed VWF pseudo-gene located on chromosome 22q11.2 which spans 25kb and corresponds to exons 23-34 of the VWF gene (15,16). Many genetic mutations and defects have been found to occur in the VWF gene which can affect different functional sites on the VWF molecules and explain the heterogeneous nature of the disorder (17).

VWF plays an essential role in primary and secondary haemostasis. At high shear stress and when vascular injury occurs, VWF multimer undergoes conformational changes that causes binding to different ligands in the sub-endothelial matrix and promotes platelet adhesion by interaction with the platelet glycoproteins Ib and II b/I IIa (GPIb and GPII b/111 a) receptors (18). Subsequent to adhesion, platelets are activated, secrete the contents of their granules, and recruit additional platelets to the site of injury. Platelet aggregates then bind to fibrinogen through the GPIIb/IIIa receptor to form a platelet plug (19).

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Furthermore, VWF functions as a carrier and stabiliser for coagulation factor VIII. VWF makes a non-covalent complex with factor VIII preventing it from proteolytic degradation and clearance from circulation (21), prolonging its half-life by fivefold (20) from approximately 2 hours to an average of 12 hours (range 9-18 hours).

Classification of VWD

In 2006, The VWF Scientific Standardisation Committee of the International Society on Thrombosis and Haemostasis revised the first classification, which was published in 1994 (12). This classification was based on available laboratory testing, however, the aim of the classification was to guide the diagnosis and treatment of patients with VWD. The committee disregarded genotypes of the disease due to limited access to genetic testing. The 2006 classification dropped the restriction of VWD due to mutations in the VWF gene, which was a criterion in the previous classification. VWD is classified into three main types (Table 1): type 1 and type 3 VWD are quantitative defects and type 2 VWD is a qualitative defect. Type 1 VWD is partial deficiency in VWF whereas type 3 VWD is virtual absence of plasma VWF. Furthermore, type 2 VWD has been divided into four subtypes according to their phenotype (2A, 2B, 2M, and 2N) (12).

Type 1 VWD represents partial quantitative deficiency in VWF that results in decreased normally functioning VWF (12), which is able to mediate platelet adhesion and binds to collagen and factor VIII under normal physiological conditions. The main laboratory findings show a reduction in both VWF antigen levels and function (9). The proportion of high molecular weight multimers patterns on protein electrophoresis are normal or show subtle decrease (23).

Type 1 is the most common form of VWD and accounts for approximately 80% of all cases (24). Patients with type 1 VWD are affected variably, may have only mild symptoms and mild decreased VWF levels, whereas others are affected more severely (1). Mostly the bleeding tendency in patients with type 1 VWD is attributed to a clearance of plasma VWF protein, not to specific abnormalities in ligand binding sites (12). Type 1 VWD is considered when laboratory assays show a reduction in VWF:Ag and VWF:RCo with a ratio close to 1 and normal multimer distribution (25). Several mutations have been identified which reduce plasma VWF levels by impairing synthesis through interfering with the intracellular transport of dimeric pro-VWF, or by enhancing rapid clearance of VWF from the circulation (9,23).

Type 1 VWD is characterised by low penetrance where people with same mutations show variable clinical signs and a variable bleeding tendency and others do not show any signs or symptoms for VWD, which makes the diagnosis of type 1 VWD a challenge (25). Patients with severe and moderate type 1 VWD can be diagnosed with ease, however, patients with mild or borderline VWF assays are difficult to diagnose. This is because these patients have no symptoms or signs and have normal lives. Also, their VWF levels are wide, ranging between 50 to 200 IU/ml. This makes 5% of population falling outside that range, in particular 2.5% of this population have VWF levels of less than 50 IU/ml. This reference range makes this group of people misdiagnosed as having VWD (26).

Furthermore, genetic variations have shown a substantial effect on VWF levels, such as the ABO blood group system. VWF levels are 25-35% lower for persons with blood group O compared with other blood groups. They have VWF levels ranging from 36-157 lU/ml (26). Also, bleeding is not uncommon in this population for many other pathological conditions.

Type 3 VWD is the most severe form of the disease and is inherited as an autosomal recessive condition (7). lt refers to a virtually complete absence of the circulating plasma VWF (12). The absolute deficiency of VWF subsequently leads to a severe deficiency in coagulation Factor VIII with plasma levels ranging between 2 and 10 U/dl (27). Type 3 VWD is very rare and it has been estimated to occur in 0.1 to 5.3 per million people (27), although there is no accurate prevalence of the disease (1).

Type 2 VWD represents qualitative abnormalities of the structure and/or function of VWF (2) and it is further subclassified into 4 categories according to the type of defect on the VWF. Current available laboratory assays allows distinguishment among different subtypes of type 2 VWD. Plasma VWF antigen usually shows a mild reduction in type 2 VWD, but may be normal. However, the assays that measures VWF activity, such as VWF:RCo and VWF:CB are reduced, except in type 2N VWD. Ideally, these assays are lower than antigen. The difference between antigen and activity assays is known as VWF functional discordance and expressed by a ratio (28).

Type 2A VWD is the most common form of type 2 VWD (1) and is characterised by the absence of large multimers that may be caused by increased susceptibility to proteolysis by ADAMTS 13 or by defective multimer assembly and retention in the endoplasmic reticulum (23,25). This results in production of small multimers that are not able to bind to platelets and connective tissue, which in turn leads to a significant decline in VWF activity (VWF:RCo and VWF: CB) (12). Levels of VWF antigen and factor VIII are normal or mildly reduced (28). To confirm the diagnosis multimer gel electrophoresis shows loss of high molecular weight multimers (25). Different mutations have been shown to reduce or interfere with multimerisation and occur in regions involved in the dimer and multimer (29). These mutations can be identified on high-resolution multimer gel electrophoresis (9). On the other side, mutations that increase VWF susceptibility to proteolysis occur within or near domain A2 (29).

Type 2B VWD is a variant that is associated with increased VWF affinity to platelet GPIb (1). Large multimers are assembled in a normal fashion but straight after secretion they bind spontaneously to platelets and are cleaved by ADAMTS 13, a disintegrin-like and metalloprotease domain with thrombosponding type 1 motifs (12). This results in the production of small multimers which are not able to mediate platelets adhesion effectively and consequently preventing their adherence to the site of injury (19,30). Often patients have associated thrombocytopenia due to increased platelet consumption. Primarily, mutations that cause type 2B VWD occur within or close to domain A1 in VWF which contains the GPIb-binding site. The mutations result in conformational changes in the A1 domain which increases the affinity of VWF for platelets and thus causing gain-of-function (31,32).

Laboratory diagnosis of type 2B VWD shows similar results to type 2A VWD with VWF:Ag, VWF:RCo, VWF:CB, and multimer gel electrophoresis. To discriminate between these two variants, ristocetin induced platelet aggregation (RIPA) is used with a low dose of ristocetin to enhance platelet agglutination in the presence of gain-of-function mutations (25).

Type 2M VWD includes a qualitative variant that is characterised by a functional defect in binding to platelet GPIb receptor (12). Patients usually have mutations within the VWF A1 domain that results in a reduced affinity to platelets and subendothelial cells (loss-of-function), while maintaining a normal assembly of the high molecular weight VWF multimers (1,31). Laboratory results are similar to type 2A VWD. However, discrimination between these two variants depends on the presence of high molecular weight multimers on gel electrophoresis (31,32).

Type 2N VWD is characterised by a reduced ability of VWF to bind coagulation factor VIII (33) that results in a marked reduction in factor VIII levels due to increased clearance from circulation (1). Patients with classical type 2N VWD show significant decreased plasma levels of factor VIII:C (< 10%) (9), despite normal VWF antigen and activity while maintaining the multimer pattern as shown by electrophoresis (33). The results from an international survey showed that factor VIII:C levels are positively correlated with the VWF capacity to bind factor VIII, with factor VIII:C plasma levels varying from approximately 10 30 % (34). The confirmation of type 2N VWD may require a VWF: factor VIII binding assay, usually in a solid-phase immunoassay (12). Mutations in type 2N VWD occur within the factor VIII binding site of VWF, which lies between amino acids Ser764 and Arg1035 and spans domain D' and the N terminal part of domain D3 (31,33).

DIAGNOSIS AND LABORATORY EVALUATION OF VWD

Clinical and laboratory evaluation of any patient suspicious for a haemorrhagic disorder, including VWD, requires an investigation of personal and family clinical history, physical examination and utilisation of several laboratory tests (Figure 2) (35). However, to confirm or exclude VWD it may require repeated laboratory testing as a significant proportion of false positives and false negatives are common. There are some factors that can affect the final outcome of the assays. For instance bleeding symptoms are frequent in normal populations without specific haemostatic defects and also the limitation of the available diagnostic assays (36). Also the wide range of VWF levels and cut-off limits for normal and low levels (mean [+ or -] 2SD) leaves 2.5% of the population as having low VWF levels. People with blood group O have VWF levels approximately 25% lower than any other blood group. This group of people can be misdiagnosed (false positive) as having type 1 VWD. Furthermore, VWF is an acute phase reactant that can increase in pregnancy, stress, infection and other inflammatory conditions. In addition, the current diagnostic assays have some issues with sensitivity, specificity, and the lower limit of detection that requires repeated testing (28). Finally, some pre-analytical issues can affect the final results, such as fear of needles, particularly in children.

Laboratory evaluation of VWD begins with screening tests followed by specific confirmatory assays. In the past, and for many years, the bleeding time was used as a gold standard screening test, but its clinical use has diminished due to difficulties with standardisation, reproducibility, and lack of sensitivity and specificity (25). Furthermore, initial haemostasis laboratory evaluation includes a complete blood count to assess the platelet count, a partial thromboplastin time (APTT) and a prothrombin time (PT). Nevertheless, this testing has limited value and does not exclude or confirm VWD (25).

In 1995, Dade-Behring introduced the platelet function analyser PFA-100 as a substitute for the bleeding time and to be used as a screening test for platelet function and bleeding disorders (37). The PFA-100 is a simple, rapid device that uses whole blood to measure platelet-based coagulation function through a capillary device to mimic high shear stress conditions that occur in vivo. The instrument gives a single endpoint reading called the closure time. Two cartridges are available for use in the PFA-100, both utilise a membrane coated with collagen; moreover, one is also coated with epinephrine and the other with adenosine diphosphate (ADP). The PFA-100 has showed a good sensitivity to VWD, estimated between 70% and 90% (37,38). However, its effectiveness is reduced due to low specificity (24 - 41%) in individuals with VWF levels greater than 25 IU/dl (25). The PFA-100 also lacks specificity to VWD and shows abnormal results in a variety of other conditions (3840).

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Recommended specific laboratory assays for VWD (Table 2) include VWF antigen, VWF activity and factor VIII coagulant plasma levels (22). When indicated, supplementary assays are used to confirm the diagnosis and assist in an accurate classification which is essential in patient management. These tests include multimer assay, ristocetin-induced platelet aggregation (RIPA), and the ability of VWF to bind factor VIII, and in some selected cases genetic testing may also be indicated.

VWF antigen assay (VWF:Ag)

This assay measures the concentration of the VWF protein in the patient's plasma. A variety of immunoassay methods are used by the majority of haemostasis laboratories to evaluate VWF:Ag levels. The most widely used are enzyme-linked immunosorbent assays (ELISA), and the automated latex immunoassay utilising a monoclonal antibody against VWF (1,36). This assay does not provide any information regarding the function or structure of the VWF protein.

VWF ristocetin cofactor assay (VWF:RCo)

Historically, VWF activity was mostly measured using the antibiotic ristocetin as a cofactor, which enhances the binding of VWF to the platelet GPIb receptor (42). It is a functional assay of VWF that measures the ability to agglutinate platelets in the presence of the antibiotic ristocetin (43). Since its introduction in the early 1970s, ristocetin is considered the test of choice for measuring VWF activity (44). In vivo, the binding of platelets to VWF through interaction with platelet GPIb receptors under shear stress induces conformational changes in the VWF. However, this interaction is promoted by ristocetin under a static condition in vitro (44). Initially, the assay was performed using a platelet aggregometer, where ristocetin is added to a mixture of patient's plasma and formalin-fixed normal platelets. The slope of the agglutination curve is proportional to the amount of the VWF activity in plasma (42). Decreased VWF:RCo, in the presence of normal VWF:Ag, is indicative of dysfunctional VwF binding to GPIb (types 2A, 2B, and 2M VWD); whereas, proportional decreases in both assays are indicative of a quantitative decrease of a normal functioning VWF molecule (type1 VWD) (Table 3) (1).

The VWF:RCo assay is widely used to diagnose VWD and is accepted as the gold standard for VWF activity. Nevertheless, the VWF:RCo assay suffers various limitations and laboratory problems which include difficulty to perform, time consuming, poor reproducibility, sensitivity (43), difficult to interpret, and raises significant concerns in quality assurance surveys.

The most significant problem is its high inter- and intralaboratory coefficient of variation (CV) which has been estimated between 20-30%, particularly when VWF levels are lower than 12-15 IU/dl in some studies (25) and 20-30 IU/dl in others (45). In a large cross-laboratory study (41), VWF:RCo showed the highest intra-laboratory variability and the CV increases with a decrease in VWF levels. Furthermore, the VWF:RCo assay showed the highest lower limit of detection with approximately 20 IU/dl in samples with type 3 VWD where expected results should be extremely low or undetectable (41). Additionally, VWF:RCo is largely insensitive to acquired VWF abnormalities (46). Many factors are involved in imprecision, including variation of the donor platelets, suboptimal quality control, and standardisation of ristocetin reagents (24).

It has been reported that the VWF:RCo assay shows significant variations among races (47). African Americans have decreased ristocetin-induced platelet aggregation compared to Caucasians. The explanation of these findings is that this population group have polymorphisms in exon 28 that affects ristocetin-based assays, which leads to underestimation of the VWF function. Furthermore, a mutation in the P14675, just outside the A1 loop and in a region previously indicated in VWF -ristocetin interactions, which has been described as a ristocetin binding site, results in an apparent decrease in binding to ristocetin. Subsequently, this results in a marked reduction in the VWF:RCo assay (44). Other studies have demonstrated that substitution of the VWF proline triplet with either arginine or aspartic acid at position 1465-1467 disrupts ristocetin-induced GPIb binding (44).

VWF collagen binding assay (VWF:CB)

The VWF:CB assay measures the functional ability of large VWF multimers to bind external collagen. It is an ELISA assay in which patient's plasma is added to a collagen-coated ELISA plate and the amount of bound VWF is evaluated using a horseradish peroxide conjugated anti-human VWF antibody (48).

Since the VWF:RCo assay suffers several limitations, recent global attention has focused on VWF:CB assay as a supplementary test of VWF function (49). Since the VWF:CB assay shows less variability, better sensitivity, better LOD, and better sensitivity to high molecular weight VWF, it should improve discrimination of VWD types. Indeed, incorporation of the VWF:CB assay into the core test panel was noted to reduce diagnosis error rates by about 50% (48). Furthermore, it is considered to be sensitive to the presence or absence of high molecular weight multimers. Yet, the VWF:CB assay does not provide an advantage over VWF:RCo as it suffers some limitations. It has not been well standardised and a wide range of animals and collagen types have been used and showed significant changes in results. In a study by Favaloro et al. (50) when assessing 21 different preparation of collagen used to bind VWF, results showed wide variation in binding ability and discrimination between VWD types. The best binding was observed for type III collagen.

The VWF:Ag to VWF:CB ratio is believed to be useful in discriminating between type 1 and type 2 VWD, with ratios of < 0.7 being consistent with type 2 VWD (17,51). The most useful and advantageous collagen for this purpose was bovine type I/ III collagen and equine tendon type I/II and type III collagen. Moreover, concentration of these collagens showed a significant effect on the binding affinity (50). VWF:CB is considered to be sensitive to VWD variants with loss of large multimers. It has been suggested that a mixture of type 1 (95%) and type III (5%) collagen is best to use. However, this concept has been challenged by rare mutations located in the A3 domain characterised by a normal multimer pattern (17,52).

VWF: factor VIII binding assay

This assay allows accurate diagnosis of type 2N VWD in which VWF:Ag, VWF:RCo and VWF:CB levels are often normal but factor FIII:C is moderately to markedly reduced. The assay measures the ability of the patient's VWF to bind exogenous factor VIII. This assay is very complex and requires several steps. It starts with immobilisation of the patient's VWF on anti-VWF-coated plates followed by removal of endogenous factor VIII. Then, purified or recombinant factor VIII is added. The final step quantifies immobilised VWF and bound factor VIII (54). This complexity makes the assay limited to only a few reference laboratories which may lead to misdiagnosis of type 2N VWD in many haemostasis clinics.

Multimer analysis

Multimer analysis is carried out to demonstrate the distribution of the wide range of VWF multimers in a patient's plasma by electrophoresis, using agarose gel in the presence of sodium dodecyl sulphate. The multimers are stained with radiolabelled antibody to VWF and visualised by autoradiography or luminography.

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Multimer analysis (Figure 3) is very important in the diagnosis of VWD, in particular type 2 VWD. Medium-resolution gels allow detection of the presence or absence of all multimer sizes. Low-resolution gels evaluate the presence of different sized multimers (56). Patients with types 2A and 2B VWD demonstrate the absence of high and intermediate molecular weight multimers. All multimers are detected in types 1, 2M and 2N VWD. No multimers can be seen in type 3 vWd (55).

Ristocetin-induced platelet aggregation (RIPA)

Generally, in normal people and at low concentrations (<0.7 mg/ml), ristocetin does not induce platelet agglutination (22). However, if the interaction occurs at this level, it reflects an abnormality in VWF-GPIb interaction. RIPA is carried out in platelet-rich plasma, using a low concentration of ristocetin (usually <0.7 mg/mL, although ristocetin lots vary, resulting in the use of slightly different ristocetin concentrations). This low concentration of ristocetin does not induce VWF binding and aggregation of platelets in samples from normal persons, however, it does cause VWF binding and aggregation of platelets in samples from patients who have either type 2B VWD or mutations in the platelet VWF receptor. The latter defects have been termed platelet--type VWD or pseudo VWD, and they can be differentiated from type 2B VWD by the VWF:PB assay. At higher concentrations of ristocetin (1.1-1.3 mg/ml), RIPA will be reduced in patients who have type 3 VWD. However, the test is not sufficiently sensitive to reliably diagnose other types of VWD (55).

The VWF:PB measures the ability of VWF to bind to formaldehyde-fixed platelets using ristocetin at low concentration (< 0.6 mg/ml) (57). The next step is to detect the amount of VWF bound to platelets using a labelled monoclonal antibody. At that level of ristocetin, normal individuals and all types of VWD, except type 2B VWD, show no binding to platelets. PLT-VWD has normal VWF:PB and can be discriminated from type 2B VWD, which has increased binding level at a low ristocetin concentration.

AIM OF STUDY

Recently, a commercial, automated, immunoturbidimetric VWF activity assay, Innovance[R] VWF Ac (VWF:Ac, Siemens Healthcare Diagnostics, Germany) became available. This assay measures VWF binding to GPIb without ristocetin. To measure VWF activity in plasma, the VWF:Ac assay uses a recombinant form of the VWF receptor with two gain-offunction mutations (G233V and M239V in the GPIb receptor protein), captured on polystyrene particles that are coated with an antibody to GPIb. The aim of this study was to evaluate the performance of the VWF:Ac assay, comparing the results with the current platelet-based VWF:RCo assay and assessing the assay repeatability using a CS2100i coagulation analyser.

MATERIALS AND METHODS

Study population

This study was approved by Health Research South (University of Otago and Southern District Health Board) and informed consent was obtained from all participants (patients and healthy volunteers) and witnessed by the venepuncture staff member who bled the subject. The study population included 30 normal healthy volunteers and 30 individuals with type 1 and type 2 VWD. Patients (n= 30, 11 males/19 females, average age 43 years) had been recruited from the haemostasis clinic database at Dunedin Hospital.

Samples from 30 normal healthy volunteers (16 females/14 males, average age 44 year) were analysed. The selection criteria for healthy volunteers were no bleeding history and no current medication, regardless of sex and age.

LABORATORY METHODS

Sample collection

Blood was collected into 3.2% (0.109 M) sodium citrate vacutainer tubes (Becton Dickinson vacutainer[R], UK), where the citrate to blood ratio is 1:9. Samples were centrifuged at 2000g for 10 minutes to obtain platelet poor plasma (PPP) within one hour then frozen in aliquots at -20[degrees] if run within two weeks and -80[degrees] for spare aliquots. Samples were thawed for 5 minutes in a water bath at 37[degrees] directly before analysis.

VWF assays

VWF activity (VWF: Ac, Innovance VWF Ac[R]) and ristocetin cofactor activity VWF:RCo (BC von Willebrand Reagent[R]) both from Siemens Healthcare Diagnostics, Germany were measured according to the manufacturer protocol using the CS2100i coagulation analyser (Siemens Healthcare Diagnostics, Germany). Standard human plasma (Siemens Healthcare Diagnostics, Germany) was used for preparation of the calibration curves against the WHO standard for both assays. Two calibration curves for the VWF:Ac assay were obtained; a medium curve and a low curve for activity ranging from 15-160% and from 4-20%, respectively. Measurements were performed within routine laboratory analysis.

The Innovance[R] vWF:Ac kit contains three different reagents that are in ready to use liquid form. Reagent I is a suspension of polystyrene particles coated with anti-GPIb antibodies. Reagent II is a heterophilic blocking reagent and reagent III is the recombinant GPIb with activating mutations (68).

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The VWF:Ac assay principle (Figures 4 & 5) mimics the reaction in which vWf binds to GPIb. Latex particles are coated with an antibody against GPIb, to which recombinant GPIb is added. The addition of patient plasma induces a VWF-dependent agglutination, which is detected turbidimetrically (59).

To compare results and as a supplementary functional assay, we have done the VWF:CB assay (performed at a reference laboratory). All results showed good correlation with VWF:RCo and vWF:Ac assays (Figures 10 & 11), but one normal healthy volunteer with no family history of VWD and no bleeding tendency showed normal VWF:RCo and VWF:Ac assays but a very low VWF:CB assay. These results were repeated with a new sample and we were able to confirm our results. However, no genetic analysis was performed to confirm mutation in the collagen binding site.

Statistical analysis

Agreement between VWF:RCo and VWF:Ac was assessed using Bland-Altman plots, i.e. the difference between the values of VWF:RCo (reference method) and VWF:Ac were plotted versus their means. Statistical analysis was performed using Analyse-it software. p < 0.05 defined statistical significance.

RESULTS

The normal and patients samples exhibited good correlation between VWF:Ac and VWF:RCo (R2 = 0.97, p < 0.0001) (Figures 6 &7), with a mean bias of 2.7 IU/dl (Figures 8 &9) as assessed by Altman-Bland plots.

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DISCUSSION

The VWF activity assay is critically important in the diagnosis and accurate classification and management of patients with VWD. Historically, the VWF:RCo assay, which measures the ability of VWF to agglutinate pre-washed and fixed platelets in the presence of cofactor ristocetin, is considered the reference method. Nevertheless, the VWF:RCo assay is still problematic for most haemostasis laboratories and suffers many diagnostic problems. For instance, it is imprecise with an average CV of 20 -30%, and is insensitive, particularly with VWF levels of <30 IU/ dl, due to its lower detection limit (approximately 10-20 IU/dl) (46). In addition, the VWF:RCo assay shows a decrease in activity due to a mutation in the ristocetin binding site on the VWF domain A1 608). This mutation has no influence on normal VWF functions in vivo.

Some cross-laboratory studies (41,61,62) have confirmed these findings and showed a decrease in the number of laboratories using VWF:RCo activity and replacing it with new alternative methods. The assay precision refers to many factors, which include biological variation of the donor platelets. Furthermore, ristocetin is manufactured by only one manufacturer and suffers from significant lot-to-lot variability (63), which influences quality control, and standardisation of reagents. Moreover, it is insensitive to loss of high molecular weight multimer and acquired von Willebrand syndrome (46).

Consequently, various alternative VWF activity assays have been developed over the last few years in an attempt to overcome the limitations and improve the performance or even to replace the VWF: RCo assay; including the VWF collagen binding assay, an automated ristocetin cofactor activity assay using an automated coagulation analyser (64), a recombinant platelet GPIb-based ELISA assay (65,66), flow cytometric assays (67), the latex particle-enhanced immunoturbidimetric or chemiluminescence assay which utilises monoclonal antibody against VWF-GPIb binding site at domain A1 (2,46,68,69), and a gain-of-function GPIb ELISA assay (60). Despite these improvements in methodology and instrumentation over 25 years, with significant improvement in sensitivity and reliability, subsequent evaluation studies have shown no advantage in using these assays over the current VWF:RCo assay (43), and no other assay has been able to bring the VWF:RCo assay down from its throne.

Recently, a commercial automated immunoturbidimetric VWF activity assay, Innovance[R] VWF Ac (VWF:Ac, Siemens Healthcare Diagnostics, Germany) has become available. This assay measures VWF binding to GPIb without ristocetin. To measure VWF activity in plasma, the VWF:Ac assay uses a recombinant form of the VWF receptor with two gain-of-function mutations, captured on polystyrene particles that are coated with an antibody to GPIb. Since its introduction in 2011, the Innovance[R] VWF:Ac assay has improved the sensitivity and precision of the VWF:Rco assay (70).

The aim of this study was to evaluate the performance and agreement between the VWF:RCo and Innovance VWF:Ac. Despite different methodologies utilised by both assay, the VWF:RCo and VWF:Ac assays showed good agreement with minimal analytical bias. The comparison of the VWF:RCo and VWF:Ac assays with normal and clinical samples exhibited a good correlation, as shown in Figures 6 and 7 (R2 = 0.97, p <0.0001). Although one sample showed a slight discrepancy between RCo and Ac with no clear clinical or sample issue, we assume it may be due to assay variability (random error). In similar study on 180 samples, Lawrie et al. (71) found similar results (r=0.99) and only one sample showed a discrepancy due to haemolysis.

The VWF:Ac assay showed a very good sensitivity with a LOD of <4 IU/dl, compared to 8-10 IU/dl for the VWF:RCo assay (all results <4 IU/dl, we have used the average of 2 IU/dl for statistical purposes). One study (71) showed a LOD of 3 IU/dl. Practically, we can achieve this level by using the low calibration curve; however, and after discussion with our haematologists, we consider this difference is not clinically significant.

Traditionally, the RCo/Ag ratio is used to guide in the discrimination between type 1 and type 2 VWD and a ratio <0.7 indicates a qualitative defect. Unfortunately, we did not perform the VWF:Ag assay (due to funding limitations) and consequently we were not able to evaluate this ratio and review our patients classification . However, previous studies (58) have shown a good performance for the VWF:Ac assay in this aspect.

Like any other turbidimetric assays, the VWF:Ac assay could theoretically be compromised by several analytical factors, such as interference with icteric, lipaemia, rheumatoid factor, haemolysis, and some specific medications. Unfortunately, we were unable to assess any such interference. To date studies (71,72) have reported a discrepancy between WF:RCo and WF:Ac due to the haemoglobin content and the presence of heterophil antibodies. Recently, Patzke J et al. (73), who developed the Innovance WF:Ac assay, have confirmed an excellent correlation with the WF:RCo assay (r = 0.99).

Our study suffered from some limitations, such as that we have not evaluated the WF:Ac assay for acquired von Willebrand syndrome and its satisfactory use for monitoring of VWF post-treatment with DDAVP (desmopressin) and/or VWF concentrate. Also, we were not able to include all subtypes of VWD. One of the main drawbacks of our study is that we were not able to perform a full panel of assays to confirm our patients' classification or, if required, reclassify them.

In conclusion, the Innovance[R] VWF:Ac assay seems to be desirable for assessing VWF activity with low levels of imprecision and improved sensitivity at low concentrations, despite rare discrepant results. Further, the simplicity using reagents supplied in a liquid ready to use form would also make this new reagent suitable for the diagnosis of VWD, in particular where experienced personel are not available. More experience would be needed to further assess the ability to discriminate between various types of VWD before possible replacement of the VWF:RCo assay and use of the VWF:Ac assay as a part of screening panel for diagnosis of VWD.

ACKNOWLEDGMENTS

I would like to thank Dr Hilda Mangos, my supervisor, for her valuable guidance and advice. She was available whenever I asked for help. I would also like to thank Brent Bishop, Technical Manager of the Haematology Department, Dunedin Hospital, for his support and encouragement and for reviewing this manuscript; Rhonda Lucas, Haemostasis Scientist, for her support and advice, particularly as this was a new assay in the laboratory; Denise McWhinnie and the staff from Patient Services at SCL Laboratory; and all my colleagues. Most importantly, I would like to thank Jan Parker, Operating Manager, Southern Community Laboratories, for approving and funding this project. This project was undertaken for Fellowship of the New Zealand Institute of Medical Laboratory Science (Inc.)

AUTHOR INFORMATION

Emil Wasef, BMLSc PGDipMLSc FNZIMLS, Medical Laboratory Scientist

Southern Community Laboratories, Dunedin

Correspondence: Emil.Wasef@sclabs.co.nz

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Emil Wasef

Southern Community Laboratories, Dunedin
Table 1. Classification of VWD according to the VWF Scientific
Standardisation Committee of the International Society on
Thrombosis and Haemostasis (ref 22)

Type   Description          Comments              Inheritance

1      Partial              Includes              Mostly autosomal
       quantitative         mutations causing     dominant
       deficiency of        rapid VWF             inheritance when
       VWF                  clearance             VWF <30 IU/dl.
                                                  Mutations with
                                                  levels >30 IU/dl
                                                  show variable
                                                  penetrance

2      Qualitative VWF defects

2A     Decreased VWF-       Some controversy      Mostly autosomal
       dependent            exists regarding      dominant
       platelet adhesion    classification of
       with selective       VWF mutations
       deficiency of high   associated with
       molecular weight     subtle reduction in
       multimers            high molecular
                            weight multimers

2B     Increased affinity   Should be             Autosomal
       for platelet GPIb    differentiated from   dominant
                            platelet-type
                            VWD; using
                            ristocetin-induced
                            platelet
                            aggregation or
                            genetic testing

2M     Decreased VWF-       Also includes         Autosomal
       dependent            defects of VWF        dominant
       platelet adhesion    collagen binding
       without selective
       deficiency of high
       molecular weight
       multimers

2N     Markedly             Should be             Commonly
       decreased            distinguished from    identified as a
       binding affinity     haemophilia A         heterozygote with
       for Factor VIII                            a VWF null allele
                                                  rather than a
                                                  homozygous form

3      Virtually            Results <3 IU/dl in   Autosomal
       complete             most assays           recessive
       deficiency of
       VWF

Table 2. Summary of VWF assay methods in diagnostic
laboratories (adapted from ref. 41)

Assay            Description

VWF:Ag           Measures plasma VWF protein level. Typically
                 performed by ELISA or LIA based methods.

VWF:RCo          Assesses VWF activity utilising ristocetin and an
                 agglutination assay. Is performed by platelet
                 agglutination or LIA based methods. For platelet
                 agglutination, the test can be performed using
                 an aggregometer or a coagulation based
                 instrument.

Factor VNI:C     Measures the activity of coagulation factor VIII.
                 It utilises a one-stage coagulometric method.

VWF:CB           Measures VWF activity utilising collagen.
                 Typically performed by ELISA.

Werfen-IL        Measures VWF activity using a monoclonal
activity assay   antibody binding assay, where the antibody is
                 directed against the platelet GPIb binding site of
                 VWF. Performed by an LIA based method.

Siemens          Measures VWF activity utilising a GPIb binding
Innovance        method. The system employs two gain-of-
activity assay   function GPIb mutations within a recombinant
(Inn VWF:Ac)     molecule that facilitates VWF binding.
                 Performed by an LIA based method.

Table 3. Expected values in VWD (ref. 9,53)

                           VWF:Ag
                           (IU/dl)

Normal                     50-200
Type 1    [down arrow] or [down arrow][down arrow]
Type 2A                 [down arrow]

Type 2B                 [down arrow]
Type 2M                 [down arrow]
Type 2N                       N
Type 3                     absent

                           VWF:RCo
                           (IU/dl)

Normal                     50-200
Type 1    [down arrow] or [down arrow][down arrow]
Type 2A         [down arrow][down arrow] or
            [down arrow][down arrow][down arrow]
Type 2B           [down arrow][down arrow]
Type 2M           [down arrow][down arrow]
Type 2N                       N
Type 3                     absent

                           VWF:CB
                          (IU/dl) *

Normal                     50--250
Type 1    [down arrow] or [down arrow][down arrow]
Type 2A   [down arrow] or [down arrow][down arrow]

Type 2B   [down arrow] or [down arrow][down arrow]
Type 2M                       N
Type 2N                       N
Type 3                     absent

                         Factor                       RIPA
                         VIII:C
                         (IU/dl)

Normal                   50--200                       N
Type 1              N or [down arrow]                  N
Type 2A             N or [down arrow]             [down arrow]

Type 2B             N or [down arrow]                  N
Type 2M             N or [down arrow]             [down arrow]
Type 2N         [down arrow][down arrow]               N
Type 3    [down arrow][down arrow][down arrow]       absent

                      LD-RIPA               VWF Multimer

Normal                absent                     N
Type 1                absent                     N
Type 2A               absent                  abnormal

Type 2B   [up arrow][up arrow][up arrow]      abnormal
Type 2M               absent                     N
Type 2N               absent                     N
Type 3                absent                   absent

Table 4. Laboratory results for the study population.

Group                VWF:RCo        VWF:Ac        VWF:CB
                 Mean (SD) IU/dl   Mean (SD)   Mean (SD) IU/
                                     IU/dl          dl

Normal (n=30)        96 (35)       100 (40)      126 (61)
VWD (n=30)           21 (19)        23 (20)       31 (26)

Table 5. Laboratory results and VWD types of patient group.

Patient   VWD *    VWF:Rco   VWF:Ac   VWF:CB
                    IU/dl    IU/dl    IU/dl

1         Type 1     48        48       89
2         Type 1     39        40       32
3         Type 1     23        26       33
4         Type 2      8        20       17
5         Type 1      9        5        20
6         Type 1      8        6        15
7         Type 1     57        61       77
8         Type 1      9        17       40
9         Type 1      8        11       9
10        Type 1      8        11       12
11        Type 1      8        5        20
12        Type 2     11        13       41
13        Type 1     10        2        14
14        Type 2     12        11       13
15        Type 2      8        15       85
16        Type 1      8        2        2
17        Type 2     54        76       70
18        Type 2      9        16       24
19        Type 2      8        5        5
20        Type 1     53        49       33
21        Type 2      9        18       27
22        Type 1      9        9        8
23        Type 2      9        2        2
24        Type 1     38        41       29
25        Type 1     69        61       90
26        Type 1     10        8        14
27        Type 2      8        2        7
28        Type 2      9        17       15
29        Type 1     34        36       39
30        Type 1     50        42       43

* Old hospital database classification and not enough
information to confirm these subtypes.
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