Laboratory diagnosis of congenital von Willebrand disease

Von Willebrand disease (vWD) is caused by quantitative or qualitative defects, or both, of the von Willebrand factor (vWF), a multimeric high-molecular glycoprotein (GP). Typically, it affects the primary hemostatic system, which is reflected by a mucocutaneous bleeding tendency simulating a platelet function defect. The vWF promotes its function in two ways: (1) by supporting platelet adhesion to the injured vessel wall under conditions of high shear forces and (2) by its carrier function for factor VIIIc (FVIIIc) in plasma. Because of the complexity of the disease, diagnosis of vWD is one of the most challenging of any coagulation disorder. The stepwise diagnosis of vWD includes patients and family history, screening procedures (bleeding time [BT], filter tests, platelet counts, activated partial thromboplastin time [aPTT]), confirmatory tests (vWF antigen [vWF:Ag], vWF ristocetin cofactor activity [vWF:RCo], vWF collagen-binding [vWF:CB] assay, ristocetin-induced platelet aggregation [RIPA], FVIIIc) and tests for final classification (multimeric analysis, FVIII binding capacity of vWF [vWF:FVIIIB], platelet vWF). In 1999, we classified 303 patients with congenital vWD as type 1 (n = 122), type 2 (n = 171), and type 3 (n = 10). Type 2 was further subdivided into type 2A (n = 126), type 2B (n = 17), type 2M (n = 22), and type 2N (n = 6). Type 2A showed a remarkable heterogeneity, with only 27.8% (n = 36) of the "classic" IIA pattern. The other high-frequency patterns were type IB (25.4% n = 32) and type IIE/F/H-like structural abnormalities (28.6% n = 36). The spectrum was completed with samples from patients with types 2D, 2C, 2C Miami, smeary structures, and other rare subtypes (together 18.9% n = 23).     

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

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

Laboratory diagnosis and management of von Willebrand disease in South Africa

Patients with Von Willebrand disease (VWD) in South Africa are cared for in 17 Hemophilia Treatment Centers. The exact prevalence of the disease is uncertain, but 539 patients are annotated in registries. VWD patients are mostly diagnosed in the five largest academic centers, and the classification of the subtypes is performed by one of these, the VWD testing facility. An algorithm is used for the diagnosis of VWD. The distribution of subtypes diagnosed by the VWD reference center is 38%, 58%, and 4% for type 1, 2, and 3, respectively, and ~15% of plasma samples received are rejected due to poor storage and transport conditions. A novel single nucleotide polymorphism has been found in an African patient with type 2B VWD. From the type 1 VWD patients who were diagnosed by the VWD testing facility, 45% seem to have an increased VWF clearance phenotype with a propeptide-to-antigen ratio of 1.9?±?0.3. VWD patients are treated with desmopressin, factor (F)VIII/VWF concentrate (Haemosolvate FVIII; National Bioproducts Institute, Durban, South Africa), and tranexamic acid. Haemosolvate FVIII contains a VWF antigen concentration of 167?±?27 IU/mL, a ristocetin cofactor activity of 100?±?29 IU/mL, a collagen binding activity of 99?±?29 IU/mL, normal VWF multimers, and a FVIII concentration of 50 IU/mL. Not all patients with VWD are currently classified, and many VWD patients in South Africa are probably undiagnosed.     

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

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

血管性血友病

血管性血友病(von Willebrand disease，vWD)是临床上一种常见的遗传性出血性疾病，其发病机理是患者的血管性血友病因子(von Willebrand factor，vWF)基因突变，导致血浆vWF数量减少或质量异常。vWD相当常见，但轻症患者可无出血表现，故很难准确统计其发病率。国外报道一般在千分之一至五千分之一。     

Laboratory monitoring of therapy in von Willebrand disease: efficacy of the PFA-100 and von Willebrand factor:collagen-binding activity as coupled strategies

Clinical management of von Willebrand disease (or von Willebrand disorder [vWD]) often involves factor replacement or desmopressin acetate (DDAVP) therapy to control (potential) bleeding. Laboratory monitoring involves testing patient samples prior to therapy and at discreet time points after therapy. Classical testing generally comprises assays for factor VIII:coagulant activity, von Willebrand factor (vWF):antigen and vWF:ristocetin cofactor activity. The PFA-100 (platelet function analyser) is a relatively new tool for the investigation of primary hemostasis, and studies have shown its potential utility in identifying both vWD and platelet disorders, and in monitoring DDAVP therapy in these patients. However, the PFA-100 has limited utility in monitoring factor replacement therapy. The collagen-binding activity (vWF:CB) assay is a relatively new functional vWF assay and studies have also shown its utility in identifying vWD, and in monitoring both DDAVP and factor replacement therapy in these patients. This review assesses the laboratory monitoring of therapy for vWD with a special focus on the combined potential utility of the PFA-100 and a vWF:CB assay sensitive for the presence or absence of large vWF multimers. This review should be of value to both hemostasis scientists and clinical specialists.     

Laboratory testing for von Willebrand disease: contribution of multimer analysis to diagnosis and classification

The stepwise diagnosis of von Willebrand disease (vWD) includes patient and family history, screening procedures (bleeding time, filter tests, platelet counts, activated partial thromboplastin time [aPTT]), confirmatory tests (von Willebrand factor [vWF]:antigen [Ag], vWF:ristocetin cofactor activity assay [RCo], vWF:collagen-binding test [CB], ristocetin-induced platelet agglutination [RIPA], and factor [F] VIII:coagulant activity [C]) and tests for final classification (multimeric analysis, vWF:factor VIII binding, and platelet vWF). Accumulating knowledge of the different clinical phenotypes and the pathophysiological basis of the disease have been translated into a classification that differentiates between quantitative and qualitative defects by means of quantitative and functional parameters and by analyzing the electrophoretic pattern of vWF multimers, but without inclusion of the genotype. Recently, it has been shown that with a sensitive method of multimer analysis, a > 90% genotype-phenotype relation may be achieved in the near future.     

Laboratory monitoring of replacement therapy for major surgery in von Willebrand disease

Von Willebrand disease (VWD) is an inherited haemorrhagic disorder caused by a quantitative or qualitative defect of von Willebrand factor (VWF), a multimeric plasma glycoprotein that plays a key role in platelet adhesion to the subendothelium and acts as a carrier of factor VIII (FVIII) in blood. Patients with VWD experience bleeding symptoms that are mainly localized in mucous membranes and soft tissues, and their severity depends on the degree of the primary reduction in VWF and the secondary deficiency of FVIII in plasma. Because VWD patients are also at increased risk of perioperative bleeding, a prophylactic treatment aimed to correct the dual haemostatic defect (i.e. VWF and FVIII) is warranted. This review summarizes knowledge on the current management of patients undergoing major surgery, focusing on the peri‐surgical laboratory monitoring of replacement therapy with VWF/FVIII concentrates. We suggest to monitor plasma levels of FVIII coagulant activity in the postoperative period rather than a surrogate maker of platelet‐binding VWF activity as the ristocetin cofactor assay and its recent modifications.     

von Willebrand disease: clinical and laboratory lessons learned from the large von Willebrand disease studies

During the past 25 years, our knowledge concerning the pathogenesis, diagnostic strategies, and treatment of von Willebrand disease (VWD) has increased significantly. Following the immunological differentiation of factor VIII (FVIII) and von Willebrand factor (VWF) in the 1970s and the cloning of the FVIII and VWF genes in the mid-1980s, substantial progress has been made in our understanding of this, the most common inherited bleeding disorder. We now recognize that VWD represents a range of genetic diseases all with the clinical endpoint of increased mucocutaneous bleeding. The molecular pathology of Type 2 and 3 VWD is now comprehensively documented and involves rare sequence variants at the VWF locus. In contrast, the genetic causation of Type 1 disease remains incompletely defined and in many cases appears to involve genetic determinants in addition to or instead of VWF. The diagnostic triad of a personal history of excessive mucocutaneous bleeding, laboratory tests for VWF that are consistent with VWD, and a family history of the condition remain the keystone to VWD identification. In the laboratory, measurement of VWF antigen and function continue to be the most important diagnostic studies, and while our understanding of the molecular genetic pathology of VWD has advanced considerably in the past decade, genetic testing as a component of diagnosis is limited to certain distinct subtypes of the disorder. Treatment of VWD has been relatively unchanged for the past decade and continues to involve either stimulation of the release of intrinsic VWF with desmopressin or the infusion of VWF concentrates.     

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

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

Aspects of the laboratory identification of von Willebrand disease in women

The increased prevalence of the laboratory diagnosis of von Willebrand disease (vWD) in women presenting with menorrhagia has raised concerns regarding certain specifics in vWD testing in women, including when vWD testing should be done in relation to menses and whether testing should be done while the patient is not taking an oral contraceptive (OC). These concerns have been based on historical reports that vWF and factor (F) VIII:coagulant activity levels can decrease during menses and conceivably increase the probability of diagnosing vWD when the patient is menstruating, whereas hormone therapy can increase the vWF levels and conceivably mask the diagnosis of vWD. Historically, the reports of a decrease in vWF levels during menstruation have been in a relatively small total number of patients; this has not been confirmed in two recent studies. In one study of 95 normal menstruating females sampled serially at days 4 to 7, 11 to 15, and 21 to 28, there was no variation. In another study of 40 volunteers, by cross-sectional analysis there was no difference. However, interestingly in that study, longitudinal analysis of samples showed a decrease in vWF antigen during menstruation. In another recently published study, using cross-sectional analysis, the lowest levels of vWF were found on days 1 through 4, whereas the highest were identified on days 9 through 10. Conceivably, these groups were more finely divided than were those in the other studies. In summary, in light of these conflicting results, recommendations for testing exclusively during menses cannot be made. At this point, it is not unreasonable to suggest that hematologists and gynecologists sample at least once at the time of menstrual bleeding when the diagnosis of vWD in a menstruating female is suspected. Consequently, sampling during the menstrual cycle may likely capture the lowest levels of FVIIIC and vWF antigen. Regarding testing while the patient is taking an OC, present preparations do not contain supraphysiological doses of estrogen and are unlikely to affect the laboratory diagnosis of vWD. These studies primarily have been in controls. Therefore, prospective studies in vWD women of menstrual variation of the vWF levels and the impact of OCs on vWF levels are in order before specific recommendations can be made regarding the timing of testing and whether patients should be tested while the patient is not taking an OC. Other aspects regarding race, age, preanalytical variables, and pregnancy potentially affecting the laboratory diagnosis of vWD are also discussed in this review.     

Women and von Willebrand disease

In 1926 von Willebrand made the observation that 'the trait seemed especially to be seen among women'. A pictorial bleeding assessment chart (PBAC) defines menorrhagia with a score of >100 representing >80 ml blood loss. A retrospective study has shown menorrhagia in 74% patients with von Willebrand disease (vWD) and vWD in 13% patients with menorrhagia. Although von Willebrand factor (vWF) increases in pregnancy, there may be an increased incidence of post partum haemorrhage. DDAVP is a useful treatment for menorrhagia and postnatally. Clotting factor concentrates containing vWF may be required.     

Acquired von Willebrand disease

Acquired von Willebrand disease (AvWD) is an acquired bleeding disorder which may suddenly become manifest in individuals, usually in the absence of a personal or family history of bleedings and frequently in association with monoclonal gammopathies, lymphoproliferative, myeloproliferative and autoimmune disorders. In a minority of the cases AvWD may develop in association with drugs or solid tumours. Pathogenetic mechanisms involve autoantibodies directed against von Willebrand factor (vWF) resulting in a rapid clearance of vWF from the circulation and/or inactivation of plasma vWF; absorption or adsorption of plasma vWF to malignant cells; drug-induced or cell-mediated proteolysis of plasma vWF; acquired decrease in synthesis of vWF and/or release of vWF from storage sites; or precipitation of plasma vWF. Treatment options include--whenever possible--treatment of the underlying disorder or symptomatic treatment aimed at replacing the loss of vWF by either infusion of vWF-rich concentrates or administration of desmopressin (DDAVP). In selected cases with anti-vWF antibodies, administration of high-dose intravenous gammaglobulin, plasma exchange or extracorporeal immunoadsorption may be successful.     

Acquired von Willebrand disease

Acquired von Willebrand disease (AvWD) is a syndrome that has clinical and laboratory features similar to hereditary vWD. In contrast to the latter it occurs in patients without a family history of previous bleeding tendency.     

Proteolytic processing of von Willebrand factor subunit: Heterogeneity in type-IIA von Willebrand disease

Summary Type IIA von Willebrand disease (vWD) is a heterogeneous disorder for which two different pathogenetic mechanisms have been proposed: increased proteolytic susceptibility of von Willebrand factor (vWF), and/or interference of its post-translational processing. Subunit analysis of vWF in type-IIA vWD has revealed an increased relative proportion of the 176- and 140-kDa subunit-derived fragments, suggesting an augmented fragmentation of vWF, even in the resting state. We analyzed the subunit pattern of vWF in plasma from five previously described patients with type-IIA vWD. All of them showed the above-mentioned pattern. In addition, the presence of a new band with an apparent molecular mass of 200 kDa, not described in normal individuals or in patients with vWD, was repeatedly observed in one of these patients. This patient also exhibited an abnormal vWF multimeric structure in platelets and in plasma, before and after desmopressin administration, when the blood was collected either in the presence or in the absence of proteinase inhibitors. We believe that an abnormal primary structure of vWF could be responsible for this abnormal proteolytic fragmentation pattern, as well as for the abnormal multimerization of vWF. Moreover, an abnormal susceptibility to proteolysis appears to be present, as suggested by the increase in the relative proportion of the 176-kDa fragment observed in the same patient. Future sequencing studies and genetic analysis may clarify whether there are one or two different defects related to the vWF of that patient. Our results indicate that the subunit analysis of vWF may reveal additional defects present in type-IIA vWD that may help our understanding of the pathogenesis of such disease.Supported in part by grants 90/3229, 91-92/0372, 94/1509 (FIS, INSALUD, Spain).     

Treatment of von Willebrand disease

Summary. von Willebrand disease is the most frequent of inherited bleeding disorders (1:100 affected individuals in the general population). The aim of treatment is to correct the dual defects of haemostasis, i.e., abnormal coagulation expressed by low levels of factor VIII and abnormal platelet adhesion expressed by a prolonged bleeding time. There are two main options available for the management of von Willebrand disease: desmopressin and transfusion therapy with blood products. Desmopressin is the treatment of choice in patients with type 1 von Willebrand disease, who account for approximately 80% of cases. This pharmacological compound raises endogenous factor VIII and von Willebrand factors and thereby corrects the intrinsic coagulation defect and the prolonged bleeding time in most type 1 patients. In type 3 and in the majority of type 2 patients desmopressin is not effective, and it is necessary to resort to plasma concentrates containing factor VIII and von Willebrand factor. Treated with virucidal methods, these concentrates are effective and currently safe, but the bleeding time defect is not always corrected by them. Platelet concentrates or desmopressin can be used as adjunctive treatments when poor correction of the bleeding time after concentrates is associated with continued bleeding.     

Treatment of von Willebrand disease

The bleeding tendency in von Willebrand disease (VWD) is heterogeneous and some patients with the mildest form of the disease have no significant bleeding symptoms throughout their lives. In some cases, the most difficult task for a clinician is to decide whether any treatment is actually required. However, cases with moderate to severe factor VIII (FVIII) and von Willebrand factor (VWF) deficiency usually require treatment to stop or prevent bleeding. Increasing autologous FVIII/VWF by desmopressin administration or providing normal allogeneic VWF through the infusion of plasma-derived concentrates can correct FVIII and VWF deficiencies and normalize or shorten bleeding time (BT). FVIII levels are the best predictors of soft tissue or surgical bleeding, while BT normalization, reflecting the correction of platelet-dependent functions of VWF, is considered a reliable indicator of an effective treatment of mucosal bleeding. Recombinant concentrates of FVIII are not indicated (apart from cases with alloantibodies against exogenous VWF), since they are devoid of VWF and lack its stabilizing effect on circulating FVIII. A very-high-purity concentrate of VWF has recently been made available, but its advantages over conventional concentrates containing both FVIII and VWF moieties are not obvious. The best way to select the appropriate treatment is to perform a test infusion with desmopressin in any patient with clinically significant VWD, provided that he/she has no contraindication to the compound or belongs to subtype with an anticipated lack of response (for example, type 3 VWD with FVIII/VWF lower than 5%).