Navigating the Diagnostic and Clinical Spectrum of Thrombocytopenia and Thrombocytopathy: Lessons from a Case Series.

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 2-year-old boy was brought to a tertiary care hospital by his mother and grandmother for evaluation of a bleeding disorder. He had no other significant medical history. The patient's mother and maternal grandmother shared a bleeding disorder and were concerned that the boy had inherited the same disease. The mother had previously used Humate P (40 IU/kg q8–12h) as needed for bleeding episodes. The patient's laboratory studies demonstrated a prothrombin time of 13.1 seconds (normal range, 11.9–14.1 seconds), an international normalized ratio of 1.0 (normal range, 0.9–1.2), a platelet count of 206 × 103/μL (normal range, 160–450 × 103/μL), and an activated partial prothrombin time of 34 seconds (normal range, 24–33 seconds). Additional tests showed a factor VIII activity level of 44% (normal range, 50%–150%), a von Willebrand antigen level of 29% (normal range, 50%–150%), and a von Willebrand activity of 19% (normal range, 50%–150%). A bleeding time test was not performed. Platelet aggregation studies demonstrated marked aggregation of the patient's platelets in the presence of 1.0% and 0.5% ristocetin, channels 1 (blue) and 2 (olive), respectively (Figure 1; channel 3 (red)—5 μg/mL collagen, channel 4 (green)—8 μM epinephrine). Platelet aggregation was also seen in the presence of 0.2% to 0.4% ristocetin (data not shown). The reaction of normal control platelets to 1.0% (Figure 2; channel 1, blue) and 0.5% (Figure 2; channel 2, olive) ristocetin is also shown.What is your diagnosis?von Willebrand disease is the most common inherited bleeding disorder and is usually inherited in an autosomal-dominant fashion. The dysfunction is due to a quantitative or qualitative defect in von Willebrand factor, leading to the disruption of primary hemostasis. Normally, von Willebrand factor mediates the adhesion and aggregation of platelets to the subendothelium in areas of high shear force (as found in arteries). In addition, von Willebrand factor protects factor VIIIc from inactivation by forming a complex with it.Inherited von Willebrand disease is divided into 3 main categories depending on whether the dysfunction is due to a quantitative (types 1 and 3) or a qualitative (type 2) deficiency in the factor. Type 1 is by far the most common, accounting for the majority of cases, and is the mildest form of the disease.1 In contrast, in type 3 von Willebrand disease, there is an almost total absence of von Willebrand factor. This form is very rare and the most severe.Type 2 von Willebrand disease can be further subclassified as type 2A, 2B, 2M, or 2N. Type 2A is the most common of this group and refers to qualitative variants of von Willebrand factor that express decreased platelet-dependent function associated with the absence of high-molecular-weight multimers. Type 2M (M for multimers) may involve a mutation that effectively inactivates binding sites for ligands on platelets and collagen. Thus, the multimeric distribution of von Willebrand factor is normal in patients with type 2M disease, but the binding to platelets is impaired. Type 2N (N for Normandy) resembles hemophilia A in that plasma levels of factor VIII are decreased. In contrast to hemophilia A, the low circulating levels of factor VIII in type 2N von Willebrand disease are caused by the decreased half-life of factor VIII due to its inability to bind the abnormal von Willebrand factor expressed in these patients.The patient described in this report had type 2B von Willebrand disease. Type 2 variants account for 20% to 30% of all cases of von Willebrand disease.2 As with most forms of von Willebrand disease, inheritance of type 2B von Willebrand disease is predominantly autosomal dominant; however, cases with apparent recessive inheritance patterns have been described. This subtype of von Willebrand disease is characterized by von Willebrand factor molecules that express an increased affinity for the platelet membrane glycoprotein GPIb. The multimeric structure of von Willebrand factor found in platelets and cultured endothelial cells from patients with the disorder appears normal.3 To date, at least 21 missense mutations and 1 small insertion have been identified in type 2B von Willebrand disease.2 These mutations appear to be restricted to an area containing the GPIb-binding site, the von Willebrand factor A1 domain. As can be surmised, there is a high degree of heterogeneity in type 2B disease. Patients expressing different mutations can exhibit increased platelet aggregation, thrombocytopenia, and some loss of multimers.The different subtypes of von Willebrand disease can be delineated by a good clinical and family history in conjunction with laboratory studies.4 General screening tests include a bleeding time, a platelet count, and an activated partial prothrombin time. The bleeding time is usually prolonged in von Willebrand disease. This test is not specific but will detect quantitative or qualitative changes in von Willebrand factor or platelets. Impairments in the function of the vessel wall can also be shown with a bleeding time. The value of the activated partial prothrombin time is minimal in most cases of von Willebrand disease. Where this test may be of import is for patients with low levels of factor VIIIc and for patients with type 2N disease. The total platelet count is important to obtain because it allows the exclusion of bleeding disorders related to thrombocytopenia not related to von Willebrand disease and because it can indicate the presence of a qualitative defect in von Willebrand factor, particularly in patients with type 2B disease. Other screening tests that can be used are the filter method with high shear stress and adhesion and retention tests that attempt to simulate the high shear stress present in arterioles.Tests that can confirm the diagnosis of von Willebrand disease include assays for factor VIII activity and plasma levels of von Willebrand factor antigen. In most patients with von Willebrand disease, factor VIII activity and plasma levels of von Willebrand factor are decreased. A caveat in evaluating plasma levels of von Willebrand factor is that persons with type O blood normally express lower levels of von Willebrand factor than persons with other blood types.Ristocetin cofactor activity (VWF/Rco) is a confirmatory assay that reflects the functional property of von Willebrand factor by mimicking the platelet interaction with the GPIb/IX complex. Ristocetin, a small glycopeptide antibiotic, binds to both von Willebrand factor and GPIbα, causing a von Willebrand factor–dependent platelet agglutination. This test has the same diagnostic power as von Willebrand factor antigen levels and is required to discern between patients with type 1 and 2M von Willebrand disease. Unfortunately, VWF/Rco demonstrates considerable interassay and interlaboratory variability. This has led to the use of alternative assays in an attempt to increase reproducibility. Thus, many laboratories have begun to use the von Willebrand factor/collagen-binding activity assay. This assay is capable of detecting the absence of high- and intermediate-molecular-weight von Willebrand factor. Moreover, the normalization of the von Willebrand factor/collagen-binding activity assay to the von Willebrand factor antigen demonstrates a greater difference (ie, greater sensitivity) in distinguishing between type 2A and 2B von Willebrand disease than does the normalization of VWF/Rco to the von Willebrand factor antigen. In combination with ristocetin-induced platelet aggregation and VWF/Rco, the von Willebrand factor/collagen-binding activity assay may help delineate between type 2A, 2B, and 2M as well as type 1 von Willebrand disease.Ristocetin-induced platelet aggregation is used to show a higher than normal affinity of von Willebrand factor for the platelet GPIb/IX complex. This test is particularly useful for evaluating patients with type 2B von Willebrand disease or those with platelet type von Willebrand disease (mutations in GPIb, a very rare occurrence). In this assay (as in Figures 1 and 2), varying concentrations of ristocetin (0.5, 1.0, and 1.5 mg/mL) and platelet-rich plasma are mixed. The minimal concentration of ristocetin able to cause 30% aggregation is recorded. Patients that demonstrate aggregation in 0.5 mg/mL of ristocetin usually suffer from type 2B von Willebrand disease.As mentioned, the von Willebrand factor gene is highly polymorphic. The traditional method used to analyze polymorphisms is restriction fragment length polymorphisms. These arise when a single nucleotide difference exists between genomic DNA sequences. These differences result in either the creation or destruction of restriction enzymatic sites. Southern blot tests can subsequently be used to analyze digestion differences. Polymorphisms may also be analyzed by polymerase chain reaction amplification of the region of interest, followed by restriction enzyme digestion and electrophoresis. More recent developments that may be useful in detecting mutations and polymorphisms are conformation sensitive gel electrophoresis, and denaturing high-performance liquid chromatography. In conformation sensitive gel electrophoresis, DNA heteroduplexes are detected by their migration pattern on a mildly denaturing gel. Similarly, denaturing high-performance liquid chromatography relies on the separation of heteroduplexes formed by the mixing, denaturing, and reannealing of 2 or more chromosomes using an alkylated, nonporous poly(styrene-divinylbenzene) stationary phase. The molecular methods used to diagnose bleeding disorders have been reviewed elsewhere.5There are multiple accepted therapies for the treatment of von Willebrand disease. The efficacy and appropriateness of the treatment depend on the type of disease that is present; however, the general goal of treatment is to correct coagulopathies related to low factor VIII levels and to correct prolonged bleeding times. For example, desmopressin is the therapy of choice for mild forms of von Willebrand disease. Desmopressin is counterindicated in the treatment of type 2B von Willebrand disease because of transient thrombocytopenia.6 For those resistant to desmopressin, factor VIII/von Willebrand factor concentrates is the recommended therapy. Other therapies that have been used to treat von Willebrand disease are antifibrinolytic amino acids (eg, epsilon aminocaproic acid and tranexamic acid) and estrogens.

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.

ADAMTS 13 deficiency is associated with abnormal distribution of von Willebrand factor multimers in patients with COVID-19

This guideline updates the previous guidelines1, 2 published on behalf of the British Society for Haematology (BSH) and the United Kingdom Haemophilia Centre Doctors' Organisation (UKHCDO), focussing on the laboratory components of diagnosis and monitoring. Clinical aspects will be addressed in a separate guideline. This guideline was compiled according to the BSH process at https://b-s-h.org.uk/media/16732/bsh-guidance-development-process-dec-5-18.pdf. The writing group, which comprised selected members of the BSH Haemostasis and Thrombosis Task Force (BSH HTTF), the UKHCDO Laboratory Working Party (LWP) and members of the UKHCDO Genetics Laboratory Network (GLN), produced the first draft of the manuscript. A literature search was carried out using the terms given in Table S1. The Grading of Recommendations Assessment, Development and Evaluation (GRADE) nomenclature was used to evaluate the levels of evidence and to assess the strength of recommendations. The GRADE criteria can be found at http://www.gradeworkinggroup.org and is summarised in appendix 3 of the guidance document linked above. Review of the manuscript was performed by the BSH HTTF, the BSH Guidelines Committee and the sounding board of BSH. It was circulated to members of the UKHCDO LWP and GLN, and was on the members section of the BSH website for comment. This guideline describes laboratory tests used to diagnose and monitor individuals with von Willebrand disease (VWD). Since the publication of the previous guideline,1 new functional tests have become widely available as an alternative to the ristocetin cofactor assay. This guideline also updates genetic testing rationale and highlights the American College of Medical Genetics and Genomics (ACMG) guidelines for interpretation of sequence variants3, 4 and Association for Clinical Genomic Science (ACGS) guidelines for variant interpretation.5 von Willebrand factor (VWF) is a large complex plasma glycoprotein essential for normal haemostasis. A reduction in VWF results in a bleeding disorder, as a quantitative defect in type 1 or type 3 VWD, or as a qualitative defect in type 2 VWD, or in acquired von Willebrand syndrome (AVWS). This varies in severity according to the degree of deficiency and the characteristics of VWF. The complex structure of VWF and the wide range of plasma VWF levels, in the normal population, during pregnancy, in acute illness and after exercise, make laboratory assessment and diagnosis challenging. A reduced VWF activity (<30 IU/dL) is usually associated with bleeding symptoms and is likely to be associated with a variant in the VWF gene, but these associations are less strong for reduced VWF activities between 30 and 50 IU/dL.1 VWF activity between 30 and 50 IU/dL in isolation may be insufficient to result in significant bleeding, although some individuals with VWF activity between 30 and 50 IU/dL do have significant bleeding symptoms: this is likely to reflect the interaction with additional abnormalities in the haemostatic pathway, including mild platelet defects.1 Caution should be exercised in diagnosing VWD in individuals with VWF between 30 and 50 IU/dL to avoid the burden of an unnecessary diagnosis and the hazard of failing to complete further investigations, but some of these individuals may have bleeding for which lack of VWF activity is the primary cause. VWF levels are known to increase with age6: VWF levels below 50 IU/dL normalise in approximately 43% of individuals.7 Levels increase in individuals with type 1 VWD, whereas in those with type 2 VWD, activity levels do not increase.8 Preanalytical issues affect the quality of test results9 for the diagnosis and monitoring of VWD, and are responsible for more than 70% of laboratory errors.10 With specialist coagulation testing often performed at regional facilities in the United Kingdom, such errors can also occur in the local laboratory prior to sample transport. VWF levels may be elevated during inflammation, after exercise, postoperatively and during pregnancy, potentially masking VWD.9 The collection of samples for diagnosis should be avoided at these times, and any diagnosis of VWD must be made on samples collected on two separate occasions. Detailed descriptions of sample handling for coagulation assays can be found in recent guidelines from the BSH11 and the International Council for Standardization in Haematology.12 Samples should be collected into 3.2% sodium citrate and transported to the laboratory as whole blood at ambient temperature (18–25°C).11, 12 Transportation of whole blood samples on ice leads to the precipitation of VWF and factor VIII (FVIII),13 and the storage of whole blood samples at 2–8°C can lead to time-dependent reduction in VWF and FVIII activity (FVIII:C).9, 14, 15 This results in a significant risk of normal individuals being misdiagnosed as having type 1 VWD, and of individuals with type 1 VWD being misdiagnosed as having a type 2 VWD.13, 16 Whole blood samples at ambient temperature are stable for 24–28 h for an activated partial thromboplastin time (APTT), VWF antigen (VWF:Ag) and VWF activity measured by the ristocetin cofactor assay (VWF:RCo), but only for 8–12 h for FVIII:C.17 Therefore whole blood samples for assays for VWD should reach the laboratory as soon as possible, and not more than 12 h, after sample collection. Clotted samples, and those that are under-filled or over-filled, should be rejected for analysis.11 Samples should be centrifuged at 18–25°C for 10 min at 1500–2000 g in a centrifuge that has a rotor with swing-out buckets.11 Stability data for citrated plasma that has been separated from blood cells show that FVIII:C reduces by up to 14.8% (depending on the reagent used) in 4 h after plasma separation.18 Therefore, plasma should be separated from cells and testing completed within 4 h of separation, and if testing cannot be completed within that time frame, plasma should be frozen for future testing. Although there are few data on the effects of haemolysis, icterus or lipaemia on VWF assays, results may be affected and plasma should be visually examined before testing.9-12 Haemolysis may lead to unpredictable effects in routine assays, depending on the mechanism and extent of haemolysis: in the absence of data to suggest otherwise, samples should be rejected and re-collected, unless in vivo haemolysis is suspected. Icterus may interfere with assays that measure optical density, especially if the assay uses a chromophore that has a similar colour to bilirubin: these interferences are assay specific and affected assays should not be performed when the interference is clinically significant. Lipaemia may cause issues with assays that measure optical density, especially in latex immunoassays. These interferences are assay specific and affected assays should not be performed when the interference would be clinically significant. High-speed centrifugation can be considered to remove the lipid layer prior to sample freezing.19 For plasma that is to be frozen, the sample should be stored in a screw cap polypropylene tube with an 'O'-ring. Samples should be stored below −70°C in freezers without auto-defrost cycles, although storage below −24°C can be used for short periods (up to 3 months).20 Frozen plasma shipped to another laboratory should be sent on dry ice to ensure that it remains frozen.11 Frozen plasma must not be allowed to thaw at room temperature (to avoid precipitation of VWF and FVIII), but should be thawed in a temperature-controlled water bath at 37°C with the surface of the frozen plasma at or below the surface of the water.12 For plasma volumes less than 1 mL, a thaw of 5 min is sufficient, although the time can be reduced if the sample is completely thawed before this time.11, 12, 21, 22 Samples should be mixed thoroughly by inversion prior to testing,11 as inadequate mixing can lead to a false VWD diagnosis.12, 21 In one study (using one reagent/analyser combination), repeated freeze–thaw cycles reduced the median result by more than would be expected by interassay variation for FVIII:C after three thaw cycles (−6.0% variation) and VWF:Ag after six cycles (−9.6% variation) but not for VWF:RCo (−0.8% after seven cycles)22; FVIII:C and VWF antigen and activity assays should not be performed on plasma aliquots that have already been through a freeze–thaw cycle, unless local verification of these findings shows otherwise. Assays for VWF and FVIII:C are particularly sensitive to differences in sample handling. One multicentre retrospective study showed frequent low VWF results in local laboratories that were not replicated when the individuals were retested at a specialist centre.23 A recent survey of participants in an international external quality assurance (EQA) scheme showed that a type 1 VWD sample with a median VWF:RCo of 28 IU/dL was correctly classified by 94% of participants, but a type 1 VWD sample with a median VWF:RCo of 15 IU/dL sample was only correctly classified by 78% of participants.24 To minimise these differences, consideration should be given to testing of samples at centres with expertise in performing the assays and interpreting the results, especially when confirming a diagnosis; collection of the samples at the site of testing should also be considered. Diagnosis of VWD/AVWS should be confirmed by testing on two occasions, but it should be noted that both intralaboratory imprecision and intraindividual variation have the potential to give false-normal results and false-abnormal results. However, studies have shown that a VWF antigen of >100 IU/dL excludes VWD on a single test with a negative predictive value of between 89%25 and 95%.26 Although decisions on the diagnosis of VWD are based on specific cut-offs of 30 or 50 IU/dL,1 it is a requirement of ISO1518927 to quote reference ranges with all examinations where possible. For VWF assays, reference ranges should be locally determined from 120 healthy donors using collection, processing and analysis techniques that are identical to those used for patient samples.11 Thus, if the laboratory primarily analyses frozen and thawed plasma, rather than fresh plasma, then reference ranges should be established with frozen plasma from normal donors. An alternative approach is to verify a manufacturer's range by analysis of 20–40 such plasmas and checking for concordance.11 Diagnostic and genetic laboratories should be accredited to ISO15189,27 which in the United Kingdom is overseen by the United Kingdom Accreditation Service. ISO15189 includes a requirement to participate in an accredited external quality assurance program, and these are available from UK National External Quality Assessment Scheme for Blood Coagulation for diagnostic and genetic laboratories, and Genomics Quality Assessment for genetics laboratories, among others. These cover all aspects of the diagnostic process from phenotypic testing to nucleic acid extraction and genetic analysis, to the description and classification of the variant(s) detected, and the production of an interpretative report. A flow chart for the laboratory investigation of suspected VWD and/or AVWS can be seen in Figure 1. In all cases of suspected VWD/AVWS, a full blood count (FBC) should be performed to count platelets and measure mean platelet volume (MPV). Platelet numbers and size are generally normal in all types of VWD, but in type 2B VWD individuals may have a mild thrombocytopenia or a normal platelet count with either normal-sized platelets28 or giant platelets.29 In platelet-type (pseudo) VWD (PT-VWD), individuals generally have a macrothrombocytopenia of varying degrees.30 Some individuals with essential thrombocythaemia (or other myeloproliferative neoplasms) may develop AVWS.31 A full initial investigation for suspected VWD/AVWS should be performed regardless of the platelet count. A coagulation screen including prothrombin time (PT), APTT and Clauss fibrinogen assay should be performed on individuals being investigated for a bleeding tendency. The laboratory reagent for APTT should be sensitive to FVIII:C < 30 IU/dL32: Many common reagents are suitably sensitive, but not all,33 and one study showed that a normal APTT could be seen in patient samples with FVIII:C as low as 12 IU/dL.34 Normal APTT results do not exclude a diagnosis of VWD/AVWS. A full initial investigation for suspected VWD/AVWS should be performed regardless of the APTT. The PFA-100/200 (PFA) analyser (Siemens, Germany) provides an in vitro assessment of some components of primary haemostasis under high shear conditions. Although the PFA cannot be recommended for the diagnosis of non-severe platelet disorders,35, 36 it may play a role in the diagnosis of VWD. The PFA sensitivity to VWD is 85%–90%, with almost 100% sensitivity to type 3, type 2A and PT-VWD.36 However, in type 1 VWD the PFA is normal in some cases with VWF levels <25 IU/dL,36 and can be normal or abnormal in individuals with low VWF.37 The PFA is twice as likely to be abnormal in individuals with platelet count <150 × 109/L compared to those with platelet count >150 × 109/L,38 and will also be abnormal in those with a low haematocrit (<0.25).39 A full initial investigation for suspected VWD/AVWS should be performed regardless of the results of a PFA screen. The skin bleeding time should not be used as it is poorly standardised and poorly reproducible.40, 41 While individuals with blood group O have up to 25% lower levels of VWF than non-O individuals, and therefore are more likely to have a diagnosis of type 1 VWD made,6 they are no more likely to inherit type 2 or type 3 VWD, and the bleeding phenotype of individuals with VWD is the same regardless of blood group. Therefore, there is no need for blood group-specific reference ranges,7 nor to check blood group for VWD diagnosis. FVIII:C should be measured in all individuals suspected of having VWD or AVWS. This can be measured by a one-stage clotting assay (OSCA) or chromogenic substrate assay (CSA) and numerous guidelines are available for best practice.7, 11 A VWD/AVWS diagnosis may be missed if the clinical request is for FVIII:C only,9, 10 so a FVIII:C assay alone is not enough to make or exclude a diagnosis of VWD/AVWS. VWF:Ag should be measured in all individuals suspected of VWD or AVWS. Levels are quantified by immunological methods that include enzyme-linked immunosorbent assays (ELISAs), automated immunoturbidometric methods utilising latex particle agglutination (LIA) and more recently by chemiluminescent immunoassay (CLIA) methodology.42 Limitations of LIA and CLIA assays include the presence of interfering factors such as high rheumatoid factor levels or heterophile antibodies that can falsely elevate levels.1 Furthermore, the lower level of detection and the lower limit of quantification (LLoQ) vary between assays and should be verified prior to use in each laboratory.11 Many LIA VWF:Ag methods cannot easily discriminate severe type 1 VWD from type 3 VWD due to lack of sensitivity below 2–3 IU/dL. These include the HemosIL von Willebrand Factor Antigen,43 Hyphen LIAPHEN vWF-Ag,44 Siemens VWF Ag45-47 and Stago STA-Liatest VWF:Ag48 assays. VWF:Ag assays based on flow cytometric techniques have not yet been proven to be as sensitive as automated assays.49 Pending further data, they cannot be recommended for use in the diagnosis of VWD. For the diagnosis of type 3 VWD, the assay used for measuring VWF:Ag should be demonstrably capable of measuring to <1 IU/dL. If the VWF:Ag assay used has LLoQ >1 IU/dL, laboratories should be aware that severe type 1 and type 3 VWD cannot be differentiated by that method and results should be interpreted accordingly. The platelet-based ristocetin-dependent assays for VWF activity50 (VWF:RCo) are long-established as a measure of VWF function and for use in VWD diagnosis and classification. However, they are affected by poor standardisation, poor reproducibility and poor sensitivity at low levels (although these have been partially addressed by automated assays).51-53 Alternative functional assays have been developed and automated during the last decade as surrogate measures of VWF-binding ability to platelet GPIb-V-IX, but, like VWF:RCo, they are not physiological. Assays contain either ristocetin plus wild-type recombinant GPIb-fragment or mutant recombinant GPIb-fragment containing two gain-of-function mutations with enhanced ability to bind VWF in the absence of ristocetin. An international nomenclature differentiates these assays54 with the former being denoted VWF:GPIbR and the latter VWF:GPIbM. The current WHO 6th IS (07/316) international reference preparation for VWF in plasma has incorporated VWF:GPIbR and VWF:GPIbM into the value for VWF:RCo.55 Direct comparisons of these assays have highlighted differences in sensitivities between VWF:RCo, VWF:Ab, VWF:GPIbR and VWF:GPIbM assays which potentially alter typing and subtyping of VWD, particularly in types 2A or 2M VWD,56-58 and particularly if the variant alters the reagent-binding characteristics. However, a recent meta-analysis of published data showed comparable accuracy between VWF:RCo, VWF:GPIbR and VWF:GPIbM.59 VWF activity assays based on flow cytometric techniques have not yet been proven to be as sensitive as automated VWF:GPIbR assays.49 Pending further data, they cannot be recommended for use in the diagnosis of VWD. Laboratories reporting VWF activity assays should state clearly which type of assay has been performed, using the standard nomenclature.54 Commonly used tests available in the United Kingdom with the correct assay nomenclature are shown in Table 1. A schematic diagram of the assays is shown in Figure S1. Hyphen Biomed LIAPHEN VWF:Ag [LIA] Hyphen Biomed ZYMUTEST vWF [ELISA] Siemens vWF Ag [LIA] Stago Asserachrom VWF:Ag [ELISA] Stago STA Liatest VWF:Ag [LIA] Technoclone Technozym vWF:Ag [ELISA] Werfen AcuStar von Willebrand Factor: Ag [CLIA] Werfen HemosIL von Willebrand Factor Antigen [LIA] Helena Ristocetin Cofactor [agglutination] Siemens BC von Willebrand [agglutination] Stago STA VWF:RCo [agglutination] Werfen AcuStar von Willebrand Factor: RCo [CLIA] Werfen HemosIL VWF:RCo activity assay [LIA] Hyphen Biomed ZYMUTEST vWF:CBA [ELISA] Stago Asserachrom VWF:CB [ELISA] Technoclone Technozym vWF:CBA [ELISA] Technoclone Technozym vWF:CBA Collagen Type I [ELISA] Technoclone Technozym vWF:CBA Collagen Type VI [ELISA] Werfen AcuStar von Willebrand Factor: CB [CLIA] The VWF:RCo assay is sensitive to reduction in VWF ability to interact with platelet GPIb-V-IX, but is limited by high inter- and intra-assay variation and poor accuracy at low levels. Even with automated assays, the LLoQ for VWF:RCo is 3–10 IU/dL,60, 61 which is too high to reliably differentiate between severe type 1 and type 2 VWD from type 3 VWD. studies have shown that VWF activity measured by VWF:RCo is reduced compared to levels of VWF antigen in individuals with the or these are to a in the VWF but are not associated with a bleeding In the the variant was found at a of in in in and in and is classified as has a of VWF:GPIbR assays are based on the ability of VWF to bind recombinant wild-type GPIb-fragment in the presence of and are sensitive and Some VWF:GPIbR activity assays are falsely reduced in individuals with and but are the Werfen HemosIL VWF:RCo LIA and the Werfen AcuStar VWF:RCo CLIA measure VWF:GPIbR these assays have associated with LIA and CLIA assays as assays that use VWF:GPIbR activities in individuals with type 2B and type and are not recommended for VWF:GPIbM assays are based on the ability of VWF to bind a recombinant GPIb-fragment with two gain-of-function and are sensitive and The assay not ristocetin so is to the and The automated Siemens LIA measures VWF:GPIbM and has associated with LIA assays as high VWF:GPIbM activities have been in an with AVWS with VWF:Ag and in an with a diagnosis of type 3 VWD and VWF:Ag 2 due to the presence of an interfering to be assays that use VWF:GPIbM activities in individuals with type 2B and type and are not recommended for assays do not measure VWF function and measure activities in individuals with type 2A VWD have the and in type 2M A recent also showed that these assays were also more likely to cause a of type 1 VWD as type 2 VWD. The HemosIL VWF activity LIA measures These assays are not recommended for VWF to one of to are recommended to type 2B or 2M VWD, but the of a is and the use of a that is too low or too high may risk some it is likely that such cut-offs should be method A recent meta-analysis of studies VWF levels in the diagnosis of VWD has that a of is more than a of either or It must be noted that in some individuals, reduced can be normal VWF activity and VWF:Ag this has been in type 2B and suggest that if laboratories that they are without a reference The assessment of the ability of VWF to bind to types of can the diagnosis and subtyping of and a of individuals with 2M VWD with reduced but normal can be misdiagnosed if only a VWF activity assay is performed during the initial investigation for However, in the of the writing group these are that VWF:CB assays do not need to be performed as of the initial investigation for VWD diagnosis. The of failing to these must be the on laboratories of the assays, including and with It should also be noted that not all VWF:CB assays will all abnormalities of However, laboratories not performing VWF:CB assays should samples to another laboratory if no other cause for a bleeding is to type I and/or type is performed by or CLIA although assessment of to other types may also and CLIA assays have as The use of VWF:CB assays has been to be less sensitive than assays, with some individuals with type 2A VWD being classified as having type 1 VWD if the only functional assay performed was VWF:CB assays are sensitive to a of high and have been to with VWF:RCo assays in all but type 2 A of VWF:CB to VWF:Ag may be used as an alternative to but it must be noted that the use of is less sensitive than analysis in some individuals with of to VWF antigen can be used in with VWF to between type and 2M is no international for a to type 2 VWD. cut-offs used in studies vary between and with the for type 2 VWD with analysis methods use sodium by a such as or In these the of in the can be from the recommended to on only or abnormalities of the structure using A assay has also been for use as a reproducibility and detection of abnormalities are by using of suggest a of up to with less variation is in normal or type 1 VWD VWF analysis is not only the assay for between type 2A or 2B and 2M VWD but can also be in between type 3 and low type 1 VWD or in individuals with However, it must be that results may be with some types of VWD. have been of normal in type 2B and some of linked to genetic have been in type 2M with the type normal are in type 2M with the can be seen in type and in some with type 2M Type VWD to an where there is abnormal of VWF to FVIII specific are in the FVIII of VWF low FVIII:C to with a clinical and laboratory phenotype similar to mild A but also including a low or normal diagnosis analysis and the of a reduced between and and are likely to have a of whereas some individuals may have a of of can be in normal blood donors with reference ranges should be considered although to samples is often is no international standard for and no UK or which to the in the phenotypic assays. testing is therefore recommended in with phenotypic assays unless results are clearly platelet agglutination is used as a of qualitative VWF to platelet glycoprotein Ristocetin at a high for platelet in normal samples, but can be reduced in VWD. with a lower of ristocetin for is usually but is associated with a type 2B VWD phenotype of platelet and potential This is also seen in individuals with so mixing studies using normal plasma and normal platelets may be used in individuals with ristocetin to differentiate between type 2B VWD and for platelet mixing studies can be found in Table Some with in VWF have to ristocetin but normal and no testing VWF and should be used for diagnosis of type 2B VWD or due to the limited of the diagnostic evidence from phenotypic into the the VWF from VWF and with a of 2–3 Type 1 and type 3 VWD cause a reduction of compared to VWF:Ag and detection of low levels of may differentiate severe type 1 VWD from type 3 VWD. of of are seen in normal individuals and in individuals with type 1 whereas have been in individuals with the to the of VWF in to of of are also in with type 2 VWD and in those with AVWS those with AVWS to essential thrombocythaemia to measure are not suggest when a is performed, the of VWF antigen and rather than of is performed in individuals with suspected enhanced and this should be up by genetic testing. Recommendations testing is for individuals with reduced VWF to rather than performing a assay develop in of

Dear Sir, It has been reported that approximately 30% of the genetic variations that influence von Willebrand Factor (VWF) levels in plasma are due to the ABO blood group of the individuals. The effect of ABO blood type on VWF expression has, therefore, been the subject of many studies over the years. Plasma VWF levels were reported to be significantly lower in group O individuals than in non-O individuals, which correlates with the increased risk of bleeding of the former. Explanations for the reduced levels of VWF in group O individuals range from the effect of ABO blood group on the rate of synthesis/secretion of VWF, to an effect on the survival of the protein and its clearance from plasma. The VWF cleaving protease ADAMTS-13, (the 13th member of the ADAMTS family of metalloproteases characterized by the combination of a disintegrin-like and metalloprotease with thrombospondins type 1 motif), was found to dispose of VWF physiologically by cleaving the peptide bond between tyrosine and methionine in the central A2 domain of VWF. The gene for ADAMTS-13 is located on chromosome 9q approximately 140,000 nucleotides from the ABO locus; this close proximity may also play a role in the 30% genetic variation that ABO exerts on VWF levels1. Maintaining a balance between VWF and ADAMTS-13 is crucial for blood haemostasis. While many pathological conditions are associated with an imbalance between these two proteins, several physiological factors have also been found to play a role in affecting the expression of these proteins. In this study we aimed to determine the effects of age, gender and ABO phenotype on the activity and antigenic levels of ADAMTS-13 in healthy males and females of Arab ethnicity. A hypothesis that the lower levels of VWF in group O individuals are mirrored by higher levels of ADAMTS-13 was also tested in this study. After obtaining consent, venous blood was collected into vacuum collection tubes containing sodium citrate (3.8 %, w/v)-(Becton, Dickinson and Company, New Jersey, USA), from 200 apparently healthy subjects (100 males and 100 females). All subjects were non-smokers and were undergoing a routine check-up at the time of blood collection. Standard, commercially available, enzyme-linked immunosorbent assay (ELISA) kits were used to determine levels of the studied protein according to the manufacturer’s description (Technoclone, Vienna, Austria). In order to measure VWF antigen levels, we used a sandwich ELISA with co-incubation of VWF and a secondary conjugated antibody (anti-VWF-POX) in a single step. The ADAMTS-13 antigen assay involved adding first ADAMTS-13 and then, after a washing step, a conjugate working solution containing anti- ADAMTS-13 POX. For the ADAMTS-13 activity assay, a recombinant VWF fragment was immobilised onto an ELISA plate, which encodes the A2 domain and the ADAMTS-13 cleavage site at Tyr1605-Met1606 and is tagged with S-transferase (GST)-histidine (GST-VWF73-His). After adding plasma, the residual, cleaved VWF fragment is measured by using a second monoclonal antibody [horseradish peroxidase (HRP)-conjugated monoclonal anti-N10] that recognises only the cleaved VWF fragment. The chromogenic substrate tetramethylbenzidine (TMB) was used to detect the reaction in all the assays. Since race was not a factor in this study, as all subjects were of Arab ethnicity, we focused on the effects of age, gender and ABO blood group on VWF and ADAMTS-13 levels. A non-parametric Spearman’s correlation analysis was performed to investigate the effects of age on the investigated proteins. As previously reported2, higher levels of VWF were found with older age (r=0.269, p<0.001). Given the size of the cohort, it is difficult to determine the degree to which the level changes with increasing age. Our analysis also showed that ADAMTS-13 activity decreased with age (r= −0.257, p<0.001), while ADAMTS-13 antigen levels were not affected by increasing age. It is not clear why there is this discrepancy, but the absolute difference between the two proteins appears to be small and is not likely to be of any physiological or clinical relevance (Figure 1). Whether the higher VWF antigen levels in older individuals is a consequence of lower activity of ADAMTS-13 is subject for further analysis. Figure 1 VWF antigen levels increase with age (r = 0.269), while ADAMTS-13 activity levels decrease (r= −0.257) (p 0.05). In order to determine whether gender had an influence on the findings, we compared VWF and ADAMTS-13 levels in males and females, regardless of blood group type. After controlling for age, females had significantly lower levels of VWF (p<0.001) compared to those in males (Table I). We also found that females had higher levels of ADAMTS-13 antigen (p<0.001); the combination of the results for VWF and ADAMTS-13 antigen levels could indicate that females are more prone to bleeding, but further investigation is recommended in a larger cohort to support or refute this hypothesis. Table I Comparison of ADAMTS-13 and vWF levels between males and females and between O blood group and non-O blood group subjects. Results are expressed as median (range). Eighty-one subjects (40.5 %) in our population had the O blood group and their median age was 32 years (range, 18–70 years), while 119 (59.5 %) were non O-blood group and had a median age of 33 years (range, 15–76). There was not a statistical difference in the age between the two group (p>0.05). While subjects with O blood group had significantly lower VWF antigen levels than those with non-O blood groups (p=0.003), there were no differences in ADAMTS-13 antigen and activity levels between the two groups (Table I). ADAMTS-13 levels continued to be not different when individual groups were compared (using the Kruskal-Wallis test), but VWF was significantly different (p=0.001) with levels increasing in the following order: O<A<B<AB (results not shown). After incorporating gender into the ABO blood group analysis, we found that only group O females had significantly lower VWF levels than non-O females [45% (16–275) vs 59% (18–181), p 0.05]. The lack of a difference may be related to ethnicity. It is well documented that ethnicity plays an important role in determining VWF levels3. To our knowledge, no studies on VWF and ABO blood group have previously been conducted in subjects of Arab ethnicity; hence this finding may be unique to our population although a study on a larger population is recommended as the small sample size represent a limitation to the current study. ADAMTS-13 antigen and activity levels were not different between the two groups in either gender (p>0.05). These findings suggest that whatever is causing lower levels of VWF is not related to quantitative changes in ADAMTS-13 antigen and/or activity but may be more related to structural difference in VWF protein in subjects of different blood groups. Although it is still not clear how ABO group can influence the proteolysis of VWF, it has been suggested that in group O individuals, the A2 domain (the site of VWF proteolysis by ADAMTS-13) adopts a conformation more permissive for ADAMTS-13 cleavage. A and B antigens were found to protect against VWF proteolysis, while VWF purified from group O blood has been shown to be cleaved faster by ADAMTS-13 protease2. ABO(H) sugars were also reported to affect the susceptibility of VWF to ADAMTS13 cleavage. O’Donnell et al. reported that a reduction in the number of terminal sugars on N-linked glycan increases the susceptibility of VWF to ADAMTS-13 proteolysis4. A study published in 2010 reported that the degree of sialylation modulated by ABO blood group (rather than ABO group itself) is the reason for altered proteolysis of VWF by ADAMTS-135. Here we have presented the first report on the effect of age, gender and ABO on the expression of VWF and ADAMTS-13 in healthy Arabs. VWF levels increased with age, while ADAMTS-13 activity decreased; however, despite being statistically significant, the correlation between age and these two proteins was weak. We confirmed that the levels of VWF antigen are lower in individuals with O blood group, but only in female subjects, who also had higher ADAMTS-13 antigen levels. ADAMTS-13 antigen and activity levels were not affected by ABO blood group. A more detailed analysis in a larger group of subjects is recommended.

HSCT-Associated Hepatic VOD Is Initiated With Preceding Appearance Of Unusually Large Von Willebrand Factor Multimers In Patient Plasmas

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

*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

Binding Of VWF To Type IV Collagen: An Additional Collagen Binding Mechanism Beyond Types I, III, and VI Collagen?

Patients with Bleeding Phenotype and Von Willebrand Exon 28 Polymorphism D1472H: A Retrospective Analysis At a Single Institution

Diagnosis of inherited platelet function disorders: guidance from the SSC of the ISTH

Utility of Repeat Testing in the Evaluation for Von Willebrand Disease in Pediatric Patients

Von Willebrand disease (VWD) is the most common inherited bleeding disorder with a prevalence of 0.1%, characterised by quantitative or functional deficiency of von Willebrand factor (VWF). VWD diagnosis is based on symptomology, biochemical and genetic tests, but limited laboratory resources and VWD heterogeneity still generate an important subdiagnosis gap worldwide and in our country. To identify the type and subtype of VWD in a cohort of patients with a history of excessive bleeding in Western Mexico. This prospective cohort study from 2012 to 2019 included patients with mucocutaneous bleeding or abnormal laboratory tests. A standardised questionnaire and confirmatory tests were applied: FVIII:C, VWF activity, VWF antigen, and VWF multimeric analysis. Of the 297 patients recruited, 207 (69.7%) were excluded because their values exceeded 50% in VWF activity and VWF antigen. Of those 90 remaining, 54 (18.2%) had low VWF, and only 36 patients (12.1%) were diagnosed with VWD. Among them, 17 (47.2%) had quantitative deficiencies, of whom 14 were assigned as type 1 and 3 as type 3.The remaining 19 cases were diagnosed as type 2 (52.8%): type 2A and 2B were the most frequent with 6 and 7 cases respectively; 4 cases were possible type 2M and two suggestive of 2N, however, this was not confirmed. This study highlights the challenges of VWD diagnosis using a comprehensive panel of diagnostic tests which should extend to supplemental tests of VWF:CB, VWF:FVIIIB, and sequencing the VWD gene to confirm the results from the panel assays.

Utility of Various Von Willebrant Factor Laboratory Tests in Assessment of Acquired Von Willebrant Syndrome in Patients with Aortic Stenosis