Guideline for laboratory diagnosis and monitoring of von Willebrand disease: A joint guideline from the United Kingdom Haemophilia Centre Doctors' Organisation and the British Society for Haematology

Abstract

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 at with the of the frozen plasma at or below the of the For plasma less than 1 a thaw of min is although the time can be reduced if the sample is thawed before this Samples should be by prior to as can lead to a VWD reduced the result by more than would be by for FVIII:C after thaw and after but not for after FVIII:C and VWF antigen and activity assays should not be performed on plasma that have been a unless local of these for VWF and FVIII:C are to in sample VWF results in local that not when the individuals at a specialist A recent of in an quality that a type 1 VWD sample with a of IU/dL was by of but a type 1 VWD sample with a of 15 IU/dL sample was only by of these should be given to testing of samples at with in the assays and the especially when a collection of the samples at the of testing should also be of should be by testing on two but it should be that and have the to results and have that a VWF antigen of IU/dL VWD on a test with a of between and Although on the diagnosis of VWD are on specific of 30 or 50 it is a of to with For VWF assays, should be from using and that are to those used for if the laboratory frozen and thawed than should be with frozen plasma from normal alternative is to a range by of such and for and genetic should be to which in the United Kingdom is by the United Kingdom a to in an quality and these are available from for for and genetic and Genomics for These aspects of the process from testing to and genetic to the and of the and the of an A for the laboratory of VWD can be in of a blood should be performed to and measure platelet and are normal in of VWD, but in type VWD individuals may have a mild or a normal platelet with or VWD individuals have a of individuals with essential may A for should be performed of the platelet A coagulation including time and assay should be performed on individuals being for a bleeding The laboratory reagent for should be to FVIII:C 30 are but not and that a normal be in samples with FVIII:C as as 12 results do not a diagnosis of A for should be performed of the The an in assessment of some components of primary Although the cannot be for the diagnosis of platelet it may a in the diagnosis of The to VWD is with to type type and in type 1 VWD the is normal in some with VWF levels and can be normal or in individuals with The is as likely to be in individuals with platelet to those with platelet and will also be in those with a A for should be performed of the results of a The bleeding time should not be used as it is and individuals with blood have up to levels of VWF than and are more likely to have a diagnosis of type 1 VWD are more likely to type 2 or type 3 VWD, and the bleeding of individuals with VWD is the of blood Therefore, there is for blood to blood for VWD FVIII:C should be measured in individuals of having VWD or This can be measured by a assay or assay and guidelines are available for A diagnosis may be if the is for FVIII:C 10 a FVIII:C assay is not to make or a diagnosis of should be measured in individuals of VWD or Levels are by that assays latex and more by of and assays the of such as factor levels or that can the of and the of between assays and should be prior to in cannot type 1 VWD from type 3 VWD to lack of below These the von Willebrand VWF and assays on have not been to be as as further cannot be for in the diagnosis of For the diagnosis of type 3 VWD, the assay used for should be of to the assay used has should be that type 1 and type 3 VWD cannot be by that and results should be The assays for VWF are as a measure of VWF and for in VWD diagnosis and are affected by and at levels these have been addressed by functional assays have been and during the as of to platelet are not ristocetin or two with to VWF in the absence of nomenclature these with the being and the The for VWF in plasma has and into the for of these assays have in between and assays which potentially and of VWD, in or and if the variant the a recent of published data between and VWF activity assays on have not been to be as as further cannot be for in the diagnosis of VWF activity assays should which type of assay has been using the used tests available in the United Kingdom with the assay nomenclature are in Table A of the assays is in S1. von Willebrand von Willebrand von Willebrand von Willebrand activity assay von Willebrand The assay is to reduction in VWF to with platelet but is by and and at with assays, the for is which is to between type 1 and type 2 VWD from type 3 have that VWF activity measured by is reduced to levels of VWF antigen in individuals with the or these are to a in the VWF but are not associated with a bleeding the the variant was found at a of in in in and in and is as has a of assays are on the of VWF to in the of and are and activity assays are reduced in individuals with and but are the and the measure these assays have associated with and assays as assays that activities in individuals with type and type and are not for assays are on the of VWF to a with two and are and The assay not ristocetin is to the and The and has associated with assays as activities have been in an with with and in an with a diagnosis of type 3 VWD and 2 to the of an to be assays that activities in individuals with type and type and are not for assays do not measure VWF and measure activities in individuals with type VWD have the and in type A recent also that these assays also more likely to cause a of type 1 VWD as type 2 The VWF activity These assays are not for VWF to of to are to type or VWD, but the of a is and the of a that is or may risk some it is likely that such should be A recent of VWF levels in the diagnosis of VWD has that a of is more than a of or It must be that in some reduced can be normal VWF activity and this has been in type and suggest that if that are without a The assessment of the of VWF to to of can the diagnosis and of and a of individuals with VWD with reduced but normal can be misdiagnosed if only a VWF activity assay is performed during the for in the of the writing these are that assays do not to be performed as of the for VWD The of failing to these must be the on of the assays, including and with It should also be that not assays will abnormalities of not assays should samples to another laboratory if cause for a bleeding is to type type is performed by or although assessment of to may also and assays have as The of assays has been to be less than assays, with some individuals with type VWD being as having type 1 VWD if the only functional assay performed was assays are to a of and have been to with assays in but type 2 A of to may be used as an alternative to but it must be that the of is less than in some individuals with of to VWF antigen can be used in with VWF to between type and is for a to type 2 used in between and with the for type 2 VWD with sodium by a such as or these the of in the can be from the to on only or abnormalities of the structure using A assay has also been for as a and of abnormalities are by using of suggest a of up to with less is in normal or type 1 VWD VWF is not only the assay for between type or and VWD but can also be in between type 3 and type 1 VWD or in individuals with it must be that results may be with some of have been of normal in type and some of linked to genetic have been in type with the type normal are in type with the can be in type and in some with type VWD to an there is of VWF to FVIII specific are in the FVIII of VWF FVIII:C to with a and laboratory similar to mild A but also including a or normal diagnosis 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 with should be considered although to samples is often is for and or which to the in the testing is in with assays unless results are platelet is used as a of qualitative VWF to platelet glycoprotein at a for platelet in normal samples, but can be reduced in with a of ristocetin for is usually but is associated with a type VWD of platelet and This is also in individuals with using normal plasma and normal may be used in individuals with ristocetin to between type VWD and for platelet can be found in Table with in VWF have to ristocetin but normal and testing VWF and should be used for diagnosis of type VWD or to the of the evidence from into the the VWF from VWF and with a of 1 and type 3 VWD cause a reduction of to and of levels of may type 1 VWD from type 3 of of are 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 those with to essential to measure are not suggest when a is the of VWF antigen and than of is performed in individuals with and this should be up by genetic testing. Recommendations testing is for individuals with reduced VWF to than a assay in of individuals with type 3 and can be associated with for in and a of The are is laboratory assay for diagnosis of VWF are often although there is in depending on which assay is to measure VWF testing may be the in the individuals with by this also by and only by do not time or temperature and the of the samples can be performed at for between 15 min and 2 with results in should be performed in an laboratory with the assay. may be if the are The of is complex and not to in those there is an an that to VWF and VWF without VWF activity cannot be by this are an but are not available and not Therefore the absence of a not a diagnosis of a VWD diagnosis is on and laboratory genetic can or a and the of is diagnosis may an alternative diagnosis when there are and the of remains for published VWF with and within and between insufficient data from additional affected and members can the of the of of 3 and type VWD whereas type 1 and type 2 VWD Although in VWD is of affected have been as and not found to the variant or but the variant at a reduced in blood and cells The data from have to a of within VWF being is not with of may make a variant as or are to new and associated and laboratory to The VWF (VWF) is on the short of 12 It of of with being the The a which for a It of a a and a VWF with a structure VWF has a partial which is on the of with a to of with a large of as or of these the genetic and interpretation of within individuals with reduced VWF. With laboratory assays for VWD should usually be performed before to VWF although may the functional and and within this have been found in of type 2 of this can be in these must be considered a variant has not been in as the of within VWF may to be A for the laboratory of VWD can be in It be that there are to these to the of the of genetic data in the of should be when a VWF variant for within have been found at in the and are risk for VWF activity in some assays, but are not associated with a bleeding when in Therefore, if an has a VWF variant that is as likely or a variant of should be for haemostatic the Genomic that VWF genetic testing may be by or Clinical by or and data should be with to in of an must be of the and of the used and the time for an are a range of available for the genetic of and the of will depending on the available to the genetic testing the United Kingdom and the of the essential of VWF is usually by or by The of the VWF should the of the including the and the and must be made to the of the when assays for of VWF. results of genetic testing should be by testing of the This may be a of the assay or by using a or by testing a in the assay or used should be in the the of and has been used to large of associated with bleeding and platelet for in the to be for an for VWF genetic the data for VWF can be from a and can and large are available from VWF by it may be that some sequence to the VWF and in may be in for data this is the for the in to of VWF that is to the should be using a to ensure the of the variant to VWF. may not be to sequence to specific of the gene, such as in VWF. should be by to the of large or of from a to the gene, may be performed using such as or using the should be to for of as by of the assay. are available for of VWF. should be by of the of VWF must be performed by specialist with the to and the of any to the of the in VWF should be according to 4 and guidelines for variant interpretation.5 VWF should be using Society to ensure of variant The sequence and used for should be in the to ensure that can be and if there are future to the The sequence used for VWF is such as the VWF variant and the variant can be to a variant has been and should be used with as may have been prior to the in of VWD on VWF activity some may not be as being associated with some may have been as prior to the of the guidelines for variant Table VWF with a that are as having data on this is not and are being for with of the VWF from the for can be used to the of the within the structure and assess the likely of a VWF laboratory should be and the and Society of laboratory and data are to of a specific a and should the of the may in this of and to individuals with a diagnosis should be for on the Haemophilia and a bleeding The of in the of VWD is and in the previous UKHCDO The to in FVIII and VWF levels varies widely such that a of is in individuals with type type type and type VWD prior to For individuals with VWD, the monitoring of FVIII:C or VWF activity and should be measured at are data the monitoring of to using or the assays have been in VWF and the to measure VWF activity and as functional in monitoring to VWF activity assay or or to be to monitor to VWF may be plasma with FVIII in and with or without normal of or The of VWF are using the is by the of FVIII:C or and VWF is a of data on the of or assays in monitoring of VWF should be performed and published on VWF but more data are should local using or if these assays are to be the assays have been in VWF and the to measure VWF activity and as functional in monitoring VWF with plasma to International for FVIII and VWF should be used for monitoring VWF unless there is specific evidence to the VWF activity assay or or to be performed to measure to VWF for should be avoided during pregnancy, and are not individuals with known VWD may monitoring during FVIII:C or VWF activity and should be measured in these VWF activity assay or or to performed to monitor VWF levels in The diagnosis of VWD in and is by to the in a and or sample and the increase in VWF when testing is clinically a deficiency or type 2 disease should be these a including a and FVIII:C or VWF activity or or and should be and results to local is likely to be for published can be considered if similar otherwise, to a specialist that can may be and levels those in by of but levels than levels up to 1 of testing may be to to the writing of the and of The to of Science and for in the literature and and for with the of the guideline. The BSH HTTF, LWP and members not in the writing at the time of writing this guideline (BSH and The would to the BSH sounding board and the BSH Guidelines for in this guideline. The BSH the during the writing of this The BSH the during the writing of this have made a of to the BSH and Task Force which may be on The have of and and and and and and and and have of to of the writing will the writing if any new evidence available that would the strength of the made in this document or it The document will be by the Task Force and the literature search will be 3 to search for any new evidence that may have been The document will be and from the BSH guidelines website if it new are an will be published on the BSH guidelines website the and in this guidance is to be and at the time of to the the BSH the any for the of this S1. The is not responsible for the or of any by the than should be to the for the

Topics & Concepts