Von Willebrand Disease and von Willebrand Factor: Laboratory Standards and Methods Regarding von Willebrand Factor Activity Assays, von Willebrand Factor Propeptide Assays, and Multimer Methodology.

In 1926, Erik von Willebrand, a Finnish internist andacademic, evaluated and described a 5-year-old girlwith extreme bleeding and bruising due to what hadbeen designated the A˚landic haemorrhagic diseaseby inhabitants of this archipelago island in the Gulfof Bothnia. Hjordis, the propositus, was the ninth of12 children born to a pedigree in which four femalesiblings had already died before the age of 4 withuncontrolled haemorrhage and in which 23 of 66family members, predominantly females, had expe-rienced significant bleeding and bruising complica-tions [1]. In fact, Hjordis herself eventually died atthe age of 13 during her fourth menstrual cycle.Professor von Willebrand mistakenly concluded thatthis bleeding diathesis was an unusual form ofhaemophilia and decided to label the new diseaseas pseudo-haemophilia to differentiate it from thesex-linked recessive haemophilia A. Little did vonWillebrand realize that this disease, eventually tobecomeeponymous[vonWillebranddisease(VWD)],would become the most commonly diagnosed con-genital bleeding disorder, with a prevalence rangingbetween 1 per 10 000 individuals to 1.3% [2]. Type1 VWD is the most common subtype, representingup to 75–80% of all cases while the subtype 3 VWDoccurs in approximately 1 per million population inthe United States and Europe [2]. Determination ofthe exact prevalence of VWD is hindered somewhatby the heterogeneity of clinical and diagnosticlaboratory features. Even Professor von Willebrandappreciated the challenges of diagnosing VWD as hecollaborated with Professor Rudolf Ju¨gens at theBerlin University to examine patients blood sampleswith a newly invented kapilla¨rtrombometer appa-ratus [3]. Although they were technically mistakenwhen they attributed the bleeding manifestations ofVWD to a platelet defect, their observations wereremarkably prescient since it was not until early1970s that the existence of a specific von Willebrandfactor (VWF) glycoprotein separate from factor VIIIwas finally appreciated and demonstrated to supportplatelet adhesion to the subendothelial matrix ofdamaged blood vessels.Over the last 81 years since von Willebranddescribed the bleeding disorder, there have beennumerous attempts and approaches to refine thediagnosis of the disease so that appropriate andefficacious treatment can be delivered. The clinicalphenotype of VWD includes, in part, information onwhether bleeding is spontaneous or related to surgi-cal or physical trauma; family history and inheri-tance pattern; menstrual history in women; age ofonset of bleeding (to distinguish between acquired vs.inherited coagulation disorders); and sites of bleed-ing(mucocutaneousvs.visceral,intra-articular,intra-muscular, or soft tissue locations). Subsequentlaboratory testing is necessary to exclude or confirmthe diagnosis and to further classify the subtype ofVWD. This article will focus on selected vagariesassociated with the ability to utilize clinical pheno-typic information gathered through the history andphysical examination to predict the presence ofVWD. The clinical diagnosis of VWD must be based,however, on more than physician suspicion andintuition. Thus, the need for confirmatory testing inthe laboratory. The frailties of conventional labora-tory testing for diagnosing VWD and the use of anin vitro surrogate of the bleeding time will also be

A Novel Rapid Screening Assay for the Diagnosis of the Phenotypic Variants of Von Willebrand Disease (VWD)

von Willebrand disease (VWD) is a bleeding disorder caused by either quantitative (type 1 and 3) or qualitative (type 2) defects of von Willebrand factor (VWF). No single available test provides appropriate information about the various functions of VWF, and the laboratory diagnosis of VWD is based on a panel of tests, including the measurement of factor VIII coagulant activity (FVIIIC), VWF antigen levels (VWF:Ag), VWF activity as measured by the ristocetin co-factor activity (VWF:RCo), the collagen-binding activity of VWF (VWF:CB), VWF multimer analysis, ristocetininduced platelet agglutination (RIPA), the factor-VIII-binding assay of plasma VWF and VWF propeptide levels. Due to the heterogeneity of VWF defects and the variables that interfere with VWF levels, a correct diagnosis of types and subtypes may sometimes be difficult, but is very important for therapy. Furthermore, the RCo assay and the RIPA test are based on platelet agglutination in reaction with the non-physiological antibiotic ristocetin. These tests also have low sensitivity and are difficult to standardise. Therefore, several analyses (tests) are required to diagnose VWD and it is important to be aware of the pitfalls to which these tests are subjected in terms of the diagnosis. In this article, the laboratory diagnosis of patients with type 1, 2A, 2B, 2M, 2N and 3 VWD will be explained by using a modified algorithm that was first proposed by the guidelines for diagnosis and treatment of VWD in Italy.

*Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden; Department of Thrombosis and Hemostasis, C2-R Einthoven Laboratory for Experimental Vascular Medicine, LeidenUniversity Medical Center, Leiden, The Netherlands; Centre for Haematology, Imperial College London, HammersmithHospital Campus, London;§INSERM U770, Cedex, France; and –Department of Pathology and Molecular Medicine, QueensUniversity, Kingston, ON, Canada

Evaluation of a von Willebrand factor three test panel and chemiluminescent-based assay system for identification of, and therapy monitoring in, von Willebrand disease

von Willebrand factor collagen binding assay with a commercial kit using type III collagen in von Willebrand disease type 2.

Most commonly inherited bleeding disorder, first described in Aland Islands by Erik von Willebrand. It occurs as a result of decrease in plasma levels or defect in von Willebrand factor which is a large multimeric glycoprotein. Monomers of this glycoprotein undergo N-glycosylation to form dimers which get arranged to give multimers. Binding with plasma proteins (especially factor VIII) is the main function of von Willebrand factor. The disease is of two forms: Inherited and acquired forms. Inherited forms are of three major types. They are type 1, type 2, and type 3; in which type 2 is sub-divided into 2A, 2B, 2M, 2N. Type 1 is more prevalent than all other types. Mucocutaneous bleeding is mild in type 1 whereas it is mild to moderate in types 2A, 2B, and 2M. Type 2N has similar symptoms of haemophilia. The pathophysiology of each type depends on the qualitative or quantitative defects in von Willebrand factor. The diagnosis is based on von Willebrand factor antigen, von Willebrand factor activity assay, FVIII coagulant activity and some other additional tests. Results should be analyzed within the context of blood group. von Willebrand factor multimer analysis is essential for typing and sub typing the disease. The management of the disease involves replacement therapy, non-replacement therapy and other therapies that include antifibrinolytics and topical agents.

Clearance mechanisms of von Willebrand factor and factor VIII.

Phenotypic Correction of von Willebrand Disease Type 3 Blood-Derived Endothelial Cells with Lentiviral Vectors Expressing von Willebrand Factor.

Many laboratories and clinicians struggle with the diagnosis of von Willebrand disease (VWD) 1, 2. VWD arises from deficiency and/or defects of von Willebrand factor (VWF), an adhesive plasma protein essential for effective primary haemostasis. VWF possesses many functional properties 3, which essentially explains the heterogeneity in clinical symptoms and bleeding risk in VWD-affected patients, as well as diagnostic challenges. Although a comprehensive panel of tests is required to diagnose VWD, and to assign a VWD type 1, 2, laboratory evaluation for the presence or absence of VWD (and other bleeding disorders) using simple screening tools has value in many situations, including urgent pre-operative testing. The Platelet Function Analyser (PFA)-100 (Siemens, Marburg, Germany) is a screening tool for primary haemostasis 4. The instrument is very easy to use, uses whole blood, and only requires 5 min per test, meaning that an assessment of a patient's primary haemostasis can be performed within less than 30 min from blood collection. The instrument measures occlusion of blood flow [so-called 'closure time' (CT)] in a shear flow environment after flowing blood is exposed to various agonists on a membrane in a cartridge device. There are currently three test cartridges in use: (i) one that contains collagen and epinephrine (C/Epi), is most sensitive to aspirin, haematocrit, platelet count, VWD and platelet dysfunction; (ii) another cartridge, which contains collagen and adenosine diphosphate (C/ADP), is relatively insensitive to aspirin, and less sensitive to VWD and platelet dysfunction; and (iii) another cartridge, which contains ADP and prostaglandin E1, is moderately sensitive to platelet P2Y12 receptor antagonists such as clopidogrel. The latter is only available in some geographical locations, and not applicable to assessment of primary haemostasis. In total, the results of the first two cartridges (C/Epi, C/ADP), being differentially sensitive to haematocrit, platelet count, anti-platelet medication, platelet function and VWF level and activity, have diagnostic implications for identification (or exclusion) of platelet defects and VWD. Indeed, sensitivity of the PFA-100 to VWD was identified 21 years ago in its first publication in 1995 5. Subsequent early publications compared the utility of the PFA-100 against the more invasive skin bleeding time (SBT), and most studies clearly identified that the PFA-100 had better sensitivity to VWD than the SBT, but perhaps less sensitivity to platelet disorders than the SBT 6-12. In a review published in 2008 4, I estimated a sensitivity of ~90% overall for VWD, with data on >700 patients, but notably, the sensitivity of the PFA-100 for severe forms of VWD was much higher. For type 3 VWD, reflecting an absence of VWF 13, sensitivity was 100%, meaning that no case of type 3 VWD was reported to yield a normal PFA-100 CT 4. Sensitivity for type 2A VWD (reflecting loss of the most functional forms of VWF, namely high molecular weight, VWF) 13 was also 100% 4. Sensitivity to other qualitative VWF defects or forms of VWD, namely 2B (reflecting hyper-adhesive VWF) 13 and 2M (reflecting dysfunctional VWF) 13, were possibly lower at around 97%, although it is also feasible that some of the published 'negative' findings instead reflected VWD 'misdiagnosis'. What cannot be argued is that the PFA-100 has reduced sensitivity to type 1 VWD 4, reflecting a quantitative deficiency of VWF 13. In these patients, the sensitivity of the PFA-100 to VWD is influenced by the level of VWF deficiency. Thus, overall sensitivity to type 1 VWD is around 80%, but sensitivity is closer to 100% for moderate to severe deficiencies (say <25 U dL−1 VWF). On the other hand, several workers have discounted the utility of the PFA-100. For example, Quiroga et al. 14 concluded both SBT and PFA-100 to be relatively insensitive to both VWD and platelet disorders, yielding respective sensitivities for the PFA of only 61.5% and 24% respectively. However, all VWD patients in this study were identified as 'type 1', and diagnostic and laboratory test data were incompletely reported. It is likely that many of these cases of 'VWD' only showed 'mild reduction' in VWF levels 15. Importantly, the current expert recommendation is to perhaps not label such patients (those with only mild reduction of VWF and with levels above ~30 U dL−1) as having VWD 16. Similarly, the platelet disorders identified by Quiroga et al. 14 were all platelet secretion defects, potentially considered among the 'mildest' of platelet disorders. There were a smattering of other papers published in the literature also questioning the clinical utility of the PFA-100 for identification of VWD. It is not my intention here to question the work of Quiroga et al. and others, but only to highlight that there appears to be a dichotomy of views – one 'camp' appears to applaud the PFA-100 and the other 'camp' laments its use. It almost seems as if we are talking about two different instruments – one 'good' and the other 'not so good'. No doubt that individual views depend on individual experiences, both with respect to the instrument used, as well as to the patient population investigated. Perhaps an analogy would be a particular make of car driven on particular roads; not all of us like the same car, and not all of us drive on the same roads. The PFA-100 celebrates its 21st birthday in 2015, with data on its use first appearing in press in 1995 5, 6. However, its history dates back a little further, having been developed from an instrument called the Thrombostat-4000. Interestingly, the first publication on the Thrombostat-4000 only appeared two years earlier, in 1993 17. However, the interest in the PFA-100 has far outweighed that of its predecessor. A recent Medline search of 'Thrombostat-4000' yielded only 23 papers; these were published in the period 1993–2001 (a total of 9 years). In contrast, the Medline search of 'PFA-100 OR PFA100' yielded 779 papers. While not all of these papers actually relate to this 'in vitro bleeding time device' [for example, the Medline search also captured phosphonoformic acid (PFA) 100 mg!], most do, and these were published in the period 1995–2015 (Fig. 1). As this represents a time span of 21 years, the PFA-100 has perhaps finally come of age, although the publication thrust does seem to be waning. It is therefore fitting that the current issue of Haemophilia includes a contribution 18 on the PFA-100 from a French group who first wrote of their initial experience with this instrument in 1998 7. The more recent publication 18 reports on 16 years of experience with the PFA-100 in the context of VWD. The authors retrospectively analysed the results (n = 6431) of 4027 patients referred to their centre between 1997 and mid-2013 in whom PFA-100 CT and VWF ristocetin cofactor (VWF:RCo) activity had been evaluated. Critically, in their experience, the PFA-100 was more effective in screening for VWF deficiency than the VWF:RCo! The negative predictive value (NPV), the positive predictive value, the sensitivity and the specificity of the PFA-100 for VWD were respectively 0.98, 0.51, 0.98 and 0.40. In other words, a normal PFA-100 CT was very effective (98%) in ruling out VWD, but a prolonged PFA-100 CT was not as useful for diagnosing VWD. The latter should not be considered a real barrier to use of the PFA-100 to screen for possible VWD. As the PFA-100 is a global test of primary haemostasis, it is (variably) sensitive to many things (as indicated before, namely: haematocrit, platelet count, anti-platelet medication, platelet function and VWF level and activity; viz. VWD). So, if a prolonged PFA-100 CT is identified, VWD is just one possibility. Indeed, in the real world, most cases of prolonged PFA-100 CT are due to low haematocrit, low platelet count, or anti-platelet medication such as aspirin. Accordingly, one should not expect any utility of the PFA-100 to rule in VWD. There are many other similar analogies in haemostasis. For example, the activated partial thromboplastin time (APTT) is sensitive to factor VIII deficiency, and thus will be abnormal in severe to moderate haemophilia A. However, the APTT is also sensitive to many other factor deficiencies (including fibrinogen, IX, XI and XII), as well as to lupus anticoagulant and also to many clinical therapy anticoagulants. Accordingly, a normal APTT may exclude a severe FVIII deficiency, but an abnormal APTT cannot be used to diagnose haemophilia A. Another analogous example is the D-dimer, where a negative D-dimer can be used (with ~98% confidence), to exclude a deep vein thrombosis (DVT); however, D-dimers are elevated in a wide variety of disease states (e.g., cancers, disseminated intravascular coagulation), as well as post surgery. Thus, a positive D-dimer has no role in 'diagnosing' a DVT. Finally, it can also be argued that the PFA-100 is similar to bleeding scores in this regard. A low bleeding score can help exclude VWD (or other bleeding disorder), but a high bleeding score cannot be used to diagnose VWD, as the diagnosis may instead be a platelet function disorder, and this diagnosis would require laboratory testing to identify. The findings reported by Ardillon et al. 18 require additional commentary. The finding that the PFA-100 CT seems better able to identify VWF deficiency (or more correctly to exclude VWF deficiency) than the VWF:RCo seems incongruous, as the former is a global assay of primary haemostasis, and the latter is a specific test of VWF activity. However, it also has to be remembered that the VWF:RCo assay is the most highly varied of the VWF assays, both in terms of methods in use (in-house vs. commercial reagents/methods, platelet aggregometry vs. automated instrument, different instruments in use) as well as in terms of reported test results 2. For example, using local external quality assessment (EQA) data, the inter-laboratory coefficient of variation (CV) for VWF:RCo averaged ~50% for samples tested in the last decade 2. In contrast, using EQA data from the same provider, inter-laboratory CVs for the PFA-100 seem to be lower, at around 20% 19. For the PFA-100, there is only the one instrument, and generally only two cartridges used for VWD testing. Although these CVs reflect inter-laboratory variation, and the data from Ardillon et al. 18 report on a single laboratory, one could propose that intra-laboratory variation, plus intra-individual variation, may also be either comparable, or perhaps even favourable for the PFA-100, depending on the method in use for the VWF:RCo. The findings reported by Ardillon et al. 18 actually closely align to our own experience, which we last reported in this journal in 2001 20. We have continued to gather prospective data on this, and plan to update our experience in press later this year. Overall, though, we would concur with the French group that in experienced hands, a reliable PFA-100 has very high NPV for exclusion of VWD (particularly with normal C/Epi). Our use of the instrument is a little different to that of the French group, and our experience also includes the supplementary use of the VWF collagen binding assay (VWF:CB) for VWD diagnosis and typing. An algorithm summarizing our current use of the PFA-100 is shown in Fig. 2. The PFA-100 is now 21 years old. Although in human terms this means it is now an adult, in instrument terms, where technology changes rapidly, this means it is relatively old, and so it has recently been 'upgraded' to the PFA-200. The PFA-200 uses essentially the same 'internal mechanics' as the PFA-100 but with (theoretically) 'enhanced' electronics (newer software, bigger screen, touch screen, etc.). However, whether the PFA-200 proves to be as effective as the PFA-100 requires separate validation. In conclusion, a good and reliable PFA instrument, in experienced hands, can be a useful screening tool of primary haemostasis. For VWD, normal PFA CTs can be used with a high degree of confidence to exclude VWD (akin to use of the D-dimer to exclude DVT). Although in the past we would have been confident to perform just a C/Epi CT for this purpose, occasional issues in 'reliability' have caused us to revise our practice to always also include a C/ADP CT. This acts in part to confirm the finding from the C/Epi, as well as providing additional information on possible defects and severity. The C/Epi is typically more sensitive to VWD than the C/ADP, and therefore VWD typically presents with prolonged C/Epi and prolonged ('moderate/severe' VWD) or normal ('mild' VWD) C/ADP. Normal C/Epi plus normal C/ADP is inconsistent with type 2A, 2B, 2M and 3 VWD, and unlikely for type 1 VWD with levels of VWF below 25–30 U dL−1. A prolonged C/ADP with normal C/Epi is unusual, and would normally lead us to repeat CT testing for confirmation. Also, like D-dimer testing for DVT, some pre-test clinical probability assessment is required to make best use of the PFA (Fig. 2). The author stated that he had no interests which might be perceived as posing a conflict or bias.

Phenotypic Variability in Carriers of Von Willebrand Factor Truncating Sequence Variants in the Zimmerman Program

von Willebrand disease (VWD) is a genetic bleeding disease due to a defect of von Willebrand factor (VWF), a glycoprotein crucial for platelet adhesion to the subendothelium after vascular injury. VWD include quantitative defects of VWF, either partial (type 1 with VWF levels <50 IU/dL) or virtually total (type 3 with undetectable VWF levels) and also qualitative defects of VWF (type 2 variants with discrepant antigenic and functional VWF levels). The most bleeding forms of VWD usually do not concern type 1 patients with the mildest VWF defects (VWF levels between 30 and 50 IU/dL). The French reference center for VWD performed a laboratory phenotypic and genotypic analysis in 1167 VWD patients (670 families) selected by their basic biologic phenotype: type 3, type 2, and type 1 with VWF levels <30 IU/dL. In these patients indeed, to achieve an accurate diagnosis of VWD type and subtype is crucial for the management (treatment and genetic counseling). A phenotype/genotype correlation was present in 99.3% of cases; 323 distinct VWF sequence variations (58% of novel) were identified (missense 67% versus truncating 33%). The distribution of VWD types was: 25% of type 1, 8% of type 3, 66% of type 2 (2A: 18%, 2B: 17%, 2M: 19%, 2N: 12%), and 1% of undetermined type. Type 1 VWD was related either to a defective synthesis/secretion or to an accelerated clearance of VWF. In type 3 VWD, bi-allelic mutations of VWF were found in almost all patients. In type 2A, the most frequent mechanism was a hyper-proteolysis of VWF. Type 2B showed 85% of patients with deleterious mutations (distinct from type 2B New York). Type 2M was linked to a defective binding of VWF to platelet glycoprotein Ib or to collagen. Type 2N VWD included almost half type 2N/3. This biologic study emphasizes the complex mechanisms for both quantitative and qualitative VWF defects in VWD. In addition, this study provides a new epidemiologic picture of the most bleeding forms of VWD in which qualitative defects are predominant.

Recognition and management of vascular lesions in von Willebrand disease

Limitations of the ristocetin cofactor assay in measurement of von Willebrand factor function.

A complete set of laboratory investigations, including bleeding time, PFA-100 closure times, factor VIII (FVIII) coagulant activity (FVIII:C), von Willebrand factor (VWF) ristocetin cofactor (VWF:RCo), collagen binding (VWF:CB), antigen (VWF:Ag) and propeptide (VWFpp), ristocetin-induced platelet aggregation (RIPA), multimeric analysis of VWF and the response of FVIII:C and VWF parameters to desmopressin (DDAVP), is necessary to fully diagnose all variants of von Willebrand disease (VWD) and to discriminate between type 1 and type 2 and between severe VWD type 1 and type 3. The response to DDAVP of VWF parameters is normal in pseudo VWD (mild VWF deficiency due to blood group O), in mild VWD type 1 and in carriers of recessive severe VWD type 1 and 3. The response to DDAVP is rather good but restricted followed by increased clearance in dominant type 1/2E, good but transient in mild type 2A group II, good for VWF:CB, with only poor response for VWF:RCo in 2M and 2U, poor in 2A group I, 2B, 2C and 2D, and very poor or non-responsive in severe recessive VWD type 1 and 3. Homozygosity or double heterozygosity for nonsense (null) mutations in the VWF gene result in recessive VWD type 3. The combination of a nonsense and missense mutation or of two missense mutations (homozygous or double heterozygous) may cause recessive severe VWD type 1. Recessive VWD type 2A subtype IIC (2C) is caused by homozygous or double heterozygous gene defects in the D1–D2 domain. Homozygosity or double heterozygosity for a FVIII binding defect of the VWF is the cause of recessive VWD type 2N (Normandy) characterized by low FVIII:C, mild or moderate VWF deficiency and normal VWF multimers. Dominant VWD type 1/2E is a mixed quantitative and qualitative multimerization defect caused by a heterozygous cysteine mutation in the D3 domain resulting in abnormal multimerization with a secretion and clearance defect of VWF not due to increased proteolysis. Dominant VWD type 1 Vicenza is a qualitative defect with normal secretion but rapid clearance with equally low levels of FVIII:C, VWF:Ag, VWF:RCo, VWF:CB and the presence of unusually large VWF multimers in plasma due to a specific mutation (R1205H) in the D3 domain. Dominant VWD type 2M and 2U are caused by loss-of-function mutations in the A1 domain resulting in quantitative/qualitative deficiencies with a selectively decreased platelet-dependent function with decreased VWF:RCo but normal VWF:CB, a relative decrease in large VWF multimers and the presence but relative loss of large VWF multimers. VWD type 2A and 2B show loss of large VWF multimers due to increased proteolysis. Dominant type 2A is caused by heterozygous missense mutations in the A2 domain. VWD type 2B is due to gain-of-function mutations in the A1 domain and differs from 2A by a normal VWF multimeric pattern in platelets and increased RIPA. DDAVP response curves and VWFpp/Ag ratios contribute to the diagnostic differentiation of VWD type 1 and 2. Rapid clearance of VWF after DDAVP with increased VWFpp/Ag ratios >10 appears to be diagnostic for VWD Vicenza. VWD type 1/2E due to the mutations in the D3 domain uniformly show increased VWFpp/Ag ratios ranging from 3.2 to 4.69 indicating clearance of the VWF/FVIII complex. Normal VWFpp/Ag ratios in mild VWD type 1 with mutations in the D1-D2 and the D4-B-C domains reflect a synthesis/secretion defect.