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Rethinking the diagnosis of von Willebrand disease
Thrombosis Research 127 Suppl. 2 (2011) S17–S21
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Thrombosis Research j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t h r o m r e s
Rethinking the diagnosis of von Willebrand disease Emmanuel J. Favaloro* Department of Haematology, Institute of Clinical Pathology and Medical Research (ICPMR), Westmead Hospital, NSW, Australia
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abstract
Keywords: von Willebrand disease VWD Diagnosis Desmopressin DDAVP Collagen binding Genetic testing
von Willebrand disease (VWD) is the most common inherited bleeding disorder and arises from deficiencies and/or defects in the plasma protein von Willebrand factor (VWF). VWD is classified into 6 different types, with type 1 identified as a (partial) quantitative deficiency of VWF, type 3 defined by a (virtual) total deficiency of VWF, and type 2 identifying four separate types (2A, 2B, 2M, 2N) characterised by qualitative defects. The classification is based on phenotypic assays including FVIII, VWF:Ag and VWF activity, typically by ristocetin cofactor (VWF:RCo), but also increasingly by collagen binding (VWF:CB). Phenotypic testing may be supplemented by multimer analysis, RIPA, and VWF:FVIII binding. Although genetic analysis is not required to diagnose VWD or to define a classification type, it may be useful in discrete situations. The current review briefly covers this diagnostic process, with a focus on newer approaches, including extended test panels and the use of data from desmopressin challenges as a diagnostic tool. © 2010 Elsevier Ltd. All rights reserved.
Abbreviations CB/Ag: DDAVP: FVIII: FVIII:C: GPIBA: HMW: LOD: RCo/Ag: RIPA: VWD: VWF: VWF:Ag: VWF:CB: VWF:FVIIIB: VWF:RCo:
collagen binding to antigen ratio desmopressin factor VIII factor VIII coagulant glycoprotein Iba high molecular weight limit of detection ristocetin cofactor to antigen ratio ristocetin-induced platelet agglutination(/aggregation) von Willebrand disease von Willebrand factor von Willebrand factor antigen von Willebrand factor collagen binding von Willebrand factor–Factor VIII binding von Willebrand factor ristocetin cofactor.
von Willebrand disease and von Willebrand factor von Willebrand disease (VWD) is the most common inherited bleeding disorder and arises from deficiencies and/or defects in the plasma protein von Willebrand factor (VWF) [1]. VWD is classified into 6 different types: type 1 (a partial quantitative deficiency of VWF), type 3 (a total deficiency of VWF), and types 2A, 2B, 2M, and 2N (characterising qualitative defects) [1] (Table 1). The * Correspondence: Dr. E. J. Favaloro, Department of Haematology, Institute of Clinical Pathology and Medical Research (ICPMR), Westmead Hospital, WSAHS, Westmead, NSW, 2145, Australia. Tel.: +61 2 9845 6618; fax: +61 2 9689 2331. E-mail address: [email protected] (E.J. Favaloro). 0049-3848 /$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
classification is based on phenotypic assays (factor VIIII [FVIII], VWF antigen [VWF:Ag] and VWF activity, usually by ristocetin cofactor [VWF:RCo] or collagen binding [VWF:CB]) [1–4]. Phenotypic testing may also be supplemented by VWF multimer analysis, ristocetin-induced platelet agglutination (RIPA), VWF:FVIII binding, and in select cases genetic analysis [1–6]. Diagnosis of VWD in an individual also requires establishing a personal history of bleeding, and this can be facilitated using a bleeding score [7]. Moreover, bleeding tendency in individuals with presumptive VWD may vary for reasons other than VWF [6]. In particular, the contribution of potential compound defects including platelet-related disorders is becoming increasing recognized [6]. Given similarities in bleeding symptoms, VWD and platelet function defects may also require application of a differential diagnostic process [8]. VWF has two main recognized functions to facilitate arrest of bleeding following injury – (i) promoting adhesion of platelets to each other and to the vasculature (‘primary hemostasis’), and (ii) binding and stabilizing FVIII (as used in ‘secondary’ hemostasis). However, VWF contains several functional binding sites in addition to those classically recognised as facilitating binding to its major platelet receptor glycoprotein Iba (GPIBA) and to FVIII, including binding sites for other platelet receptors (e.g., aIIbb3) and subendothelial matrix components such as collagen. The steadystate VWF plasma concentration and composition is also controlled by a complex process of manufacture, storage, secretion, proteolysis and clearance [1], with only some mechanisms elucidated. The heterogeneity of VWD therefore reflects the large and complex multimeric protein that VWF represents, as well as the simple fact that many factors additional to the VWF gene can influence the phenotypic presentation and an individual’s bleeding risk or clinical presentation [6].
Markedly decreased binding affinity for factor VIII
2N
Virtually complete deficiency of VWF
Decreased VWF-dependent platelet adhesion without a selective deficiency of HMW VWF multimers
2M
Rare form of VWD in developed countries (~1–5 cases per million population)
Rare form (<10%) of Type 2 VWD (~1–5 cases per million population).
Under-recognised form of Type 2 VWD.
Rare form (<10%) of Type 2 VWD (~1–5 cases per million population). Defined by enhanced responsiveness in a RIPA assay.
Most common presentation of Type 2 VWD.
Most common presentation of ‘VWD’ to laboratory, with most patients presenting with mildly reduced levels of VWF.
Comments
Typically defined by VWF levels <1% and FVIII <10%.
Defined by VWF:FVIIIB assay, with low FVIIIB/VWF ratios.
Low to normal levels of VWF, usually with VWF functional discordance detected by RCo/Ag generally <0.7, but relatively normal CB/Ag ratio. HMW VWF present, but multimers may show other abnormalities.
Low to normal levels of VWF, typically with VWF functional discordance (i.e., ratios of RCo/Ag and CB/Ag generally <0.7), loss of HMW VWF and (mild) thrombocytopenia. Atypical cases may not show this pattern.
Loss of HMW VWF. Usually low levels of VWF, with VWF functional discordance (i.e., ratios of RCo/Ag and CB/Ag typically <0.7).
Low levels of VWF, with VWF functional concordance (i.e., ratio of functional VWF/VWF:Ag approximates unity). RIPA usually normal unless VWF <20 U/dL.
Phenotypic presentation
Variable clinical response to DDAVP (some efficacy in some patients). Typically, for platelet-binding defect cases, VWF:Ag, VWF:CB & FVIII all rise (2–4× baseline), but VWF:RCo does not, with CB/Ag ratio remaining above 0.7 and RCo/Ag ratio remaining below 0.7.
Sometimes misidentified as type 1 or 2A VWD (e.g., LOD issues; assay variability, especially VWF:RCo; limited test panels).
Sometimes misidentified as (severe) type 1 VWD (e.g., LOD issues) or hemophilia A (if VWF testing not performed)
DDAVP ineffective.
Variable clinical response to DDAVP (some efficacy in some patients), depending on composite defect present.
DDAVP use contentious. DDAVP believed by some to be contraindicated, whereas others believe DDAVP represents an effective treatment in a proportion of patients. Effect of DDAVP on VWF and FVIII depends on defect. All parameters will rise initially, but may fall quickly depending on clearance mechanisms and defect.
Often misidentified as not VWD, or as type 1 VWD; sometimes misidentified as type 2A VWD (especially if VWF:CB or RIPA not performed, since VWF:Ag and/or RCo/Ag ratio sometimes normal)
Sometimes misidentified as hemophilia A/Carrier, or other forms of VWD (especially type 1)
Variable clinical response to DDAVP (some efficacy in some patients). Typically, VWF:Ag & FVIII rise (2–4× baseline), but VWF:RCo & VWF:CB do not, with CB/Ag and RCo/Ag ratios remaining below 0.7.
Usually respond well to DDAVP, unless VWF <10%. Generally, VWF:Ag, VWF:RCo, VWF:CB & FVIII all appreciably rise (2–4× baseline), with CB/Ag and RCo/Ag ratios remaining above 0.7.
Sometimes misidentified as type 2 VWD (e.g., LOD issues; assay variability, especially VWF:RCo; preanalytical issues) or misidentified as not VWD (e.g., acute phase increase in VWF due to stress, pregnancy)
Sometimes misidentified as type 1 or 2M VWD (e.g., LOD issues; assay variability, especially VWF:RCo, limited test panels)
Desmopressin (DDAVP) response profile
Phenotypic problems
Classification scheme derived and adapted from reference [1]. CB/Ag: collagen binding to antigen ratio; DDAVP: desmopressin; HMW: high molecular weight; FVIII:C: factor VIII coagulant; LOD: limit of detection; RCo/Ag: ristocetin cofactor to antigen ratio; RIPA: ristocetin induced platelet agglutination (/aggregation); VWD: von Willebrand disease; VWF: von Willebrand factor; VWF:CB: von Willebrand factor collagen binding; VWF:Ag: von Willebrand factor antigen; VWF:FVIIIB: VWF FVIII binding assay; VWF:RCo: von Willebrand factor ristocetin cofactor.
3
Increased affinity of VWF for platelet glycoprotein Ib
2B
Qualitative VWF defects
2
Decreased VWF-dependent platelet adhesion and a selective deficiency of high-molecular-weight VWF multimers
Partial quantitative deficiency of VWF
1
2A
Description
VWD type
Table 1 Classification scheme for von Willebrand disease, phenotypic presentation, phenotypic problems and desmopressin response profile
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Contribution of phenotypic characterisation to diagnosis of VWD
Contribution of phenotypic characterisation to misdiagnosis of VWD
In type 1 VWD there is a partial deficiency of VWF with similar (concordant) reductions in VWF:Ag, FVIII:C, VWF:RCo and VWF:CB. If performed, multimeric analysis would show a normal multimeric structure and RIPA would usually be normal unless levels of VWF fall below 10–20 U/dL. In type 3 VWD, VWF is (virtually) absent and all VWF tests would (in theory) yield values around 0 U/dL. If performed, multimeric analysis would show a lack of VWF and RIPA would be absent (no response to ristocetin). In type 2 VWD, VWF:Ag is usually low, but may be normal; however, functional tests will show an abnormality or reduction, with the pattern helping to identify the type [1–4]; summarised as follows: (i) In type 2A VWD, the defect is caused by a loss of high molecular weight (HMW) VWF, due either to faulty assembly or enhanced proteolysis [1]. This deficiency can be identified using multimer analysis, or by assessing VWF activity, with values of VWF:RCo and VWF:CB both lower than VWF:Ag. This test pattern is termed VWF functional discordance, and expressed numerically using ratios. Type 2A VWD is therefore typically characterised by VWF:RCo/VWF:Ag (RCo/Ag) and VWF:CB/VWF:Ag (CB/Ag) ratios below around 0.7. (ii) Type 2B VWD is characterised by enhanced binding (i.e., hyper-adhesive activity) of VWF to GPIBA, as identified by elevated RIPA responsiveness. This enhanced VWF–GPIBA binding typically causes some clearance or loss of HMW VWF (the most adhesive forms) and also VWF-bound platelets (thus mild thrombocytopenia). However, some ‘atypical’ forms of 2B VWD may not show these latter features [9,10]. Like 2A VWD, HMW VWF loss can be identified using multimer analysis, or surrogate markers such as VWF:CB and VWF:RCo. Phenotypically, then, type 2B VWD cases (like 2A) typically express RCo/Ag and CB/Ag ratios below around 0.7, particularly under periods of stress, and this pattern may lead to some 2B VWD patients being misidentified as 2A where RIPA is not performed. (iii) Type 2M VWD is recognised as an inherent VWF defect, where loss of VWF function is not due to loss of HMW VWF. Thus, although multimer analysis may show some structural abnormalities, HMW multimers will be present, and the pattern may more closely resemble type 1 VWD. Most cases of type 2M have defective binding to GPIBA; hence, VWF:RCo, which ‘functionally’ detects this binding, tends to be lower than VWF:Ag, and RCo/Ag ratios are below around 0.7. However, since collagen binding is less affected, VWF:CB values are similar to VWF:Ag (i.e., show concordance), and CB/Ag ratios are typically above 0.7. Interestingly, rare forms of 2M VWD show the opposite pattern, since the VWF defect affects collagen binding but not GPIBA binding; phenotypically this leads to low CB/Ag ratios, but normal RCo/Ag ratios [1–4,11– 13]. (iv) Type 2N VWD represents an inherent VWF defect causing defective FVIII binding. Thus, plasma FVIII is labile, prone to proteolysis, and plasma FVIII:C levels tend to be lower than VWF (i.e., low plasma FVIII/VWF ratios are evident). Phenotypically, 2N VWD tends to mimic hemophilia A or hemophilia A carriers, but can be identified using the VWF:FVIIIB assay, with low FVIIIB/VWF ratios in 2N VWD, but normal ratios in Hemophilia A (/carriers) [14].
Several major problems with phenotypic characterisation of VWD lead to ineffective diagnosis in some cases, and to misdiagnosis in others. First, there is a continuum of VWF values in all normal individuals, as well as most individuals with VWD, leading to overlaps in values between unaffected ‘normals’ and affected VWD individuals [15]. Indeed, the usual methods for establishing reference ranges for phenotype based assays (i.e. mean +/− 2 × standard deviation) by definition ascribes ~2.5% of the normal population as having a low level of VWF. Second, VWF is an acutephase reactant, and increases in times of stress, illness, infection, as well as during pregnancy. Third, many of the assays used, particularly VWF:RCo, show a high degree of assay variability, meaning different test results on different occasions in the same or different laboratories [16]. Fourth, lower limit of detection (LOD) issues are evident for many VWF assays, again in particular VWF:RCo (which may be as high as 20%) or VWF when measured by latex immuno assay (LIA) technology [17]. This poses difficulty in VWD diagnostics in particular with the most severe forms of VWD. Fifth, preanalytical issues in VWD testing can lead to identification of VWD-like patterns in normal individuals, or to incorrect VWD types being assigned [18]. Sixth, most laboratories use inappropriate or limited test panels, insufficient to properly identify phenotypically all cases of VWD. Taking these factors into consideration, and depending on presiding test panels employed and test results obtained, patients with a mild form of ‘type 1 VWD’ could easily be misidentified as not having VWD (false negative), normal individuals may be misdiagnosed as having VWD (false positive), type 3 VWD individuals may be misdiagnosed as severe type 1 or type 2 VWD, and type 1 VWD might be misdiagnosed as type 2 VWD or vice versa [19,20]. For example, LOD issues lead to poor discrimination of type 3, severe type 1 and severe type 2 VWD, either because they cannot be differentiated, or because of false concordance/ discordance by RCo/Ag. High assay variability, particularly for VWF:RCo, also means that discordance by RCo/Ag will occasionally not be observed in types 2A and 2B VWD (leading to misdiagnosis as type 1), or a false functional discordance may instead be identified (leading to misdiagnosis of type 1 as type 2A). Preanalytical issues usually cause normal individuals or type 1 VWD individuals to be misidentified as type 2 [18]. Although performance of VWF multimers might assist the diagnosis of VWD, and ‘prevent’ some of these misdiagnoses, the reality of broadly applied multimer testing is similarly not ideal. Up to 20% and 40% of laboratories will respectively identify loss of HMW VWF in normal or type 1 VWD samples (i.e., false type 2 VWD) [21]. Even ‘expert’ centres using multimer analysis also misdiagnose VWD, as around 20% of cases identified with type 1 VWD are in fact type 2 VWD, with most of these being 2M VWD [22]. This latter finding is explained by false VWF functional concordance identified due to high assay variability and LOD issues with VWF:RCo, with multimer analysis failing to resolve between type 1 and type 2M VWD. Additional useful investigative approaches to help overcome phenotypic assay limitations Diagnostic problems from phenotypic evaluation can be minimised by: (i) using improved methodologies (e.g., enzyme linked immunosorbant assay [ELISA] in place of electro-immuno diffusion [Laurel gel]), (ii) extending the test panel, (iii) repeat testing using a fresh sample for confirmation, (iv) avoiding testing of stressed, ill or pregnant patients, and (v) using data from a desmopressin (DDAVP) challenge test.
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The addition of VWF:CB to a core test panel of VWF:RCo, VWF:Ag and FVIII:C, for example, will reduce phenotypic-based diagnostic error rates by around half [19,20]. DDAVP is a non-tranfusional form of therapy given to select patients with VWD that results in release of endogenous (endothelial-cell stored) VWF. Notably, DDAVP is (nearly invariably) effective in type 1 VWD where VWF levels are above 30 IU/dL, is (usually) effective in other cases of type 1, and is sometimes effective in type 2 VWD [25]. A trial of DDAVP is commonly performed to assess clinical utility, since responsiveness is typically stable over time within individuals, despite varying between individuals. There is additional but under-recognised utility in assessing data from DDAVP trials for the diagnosis of VWD, given the response profile is often VWD-type characteristic [2,3,23]. In brief, FVIII and all VWF test parameters rise post DDAVP in type 1, and RCo/Ag and CB/Ag ratios remain above 0.7 (Fig. 1). In type 2A, FVIII and VWF:Ag rise, but VWF:RCo and VWF:CB do not, or their response is transient; therefore RCo/Ag and CB/Ag ratios typically remain below 0.7. In GPIBA-binding dysfunction type 2M, FVIII, VWF:Ag and VWF:CB typically all rise; however, VWF:RCo does not – therefore CB/Ag ratios typically remain above 0.7 but RCo/Ag ratios remain below 0.7. Thus, in patients where the VWD type is unclear, DDAVP response patterns can help assign the VWD type. The PFA-100 also has potential utility in this setting, as initially prolonged closure times will tend to shorten and normalise post DDAVP in type 1, but not in types 2A and 2M (Fig. 1; [2,3,24]). Finally, genetic testing in VWD may be of use in select investigations, namely in (i) type 2N (to help discrimination from hemophilia A/carrier); (ii) type 2B VWD (primarily for discrimination from platelet-type [PT]-VWD); (iii) type 3 VWD (for prenatal assessment/family studies and alloantibody risk assessment); and perhaps in (iv) type 2A/2M VWD and (v) type 1 VWD where VWF levels are <20–30 IU/mL [5]. In these cases, testing is focused and there may be therapeutic implications to an incorrect diagnosis (e.g., VWF concentrate vs FVIII concentrate in 2N VWD vs hemophilia; VWF concentrate vs platelet replacement in 2B VWD vs PT-VWD). However, genetic testing cannot be broadly applied to VWD diagnostics, as it typically provides only one piece of the jigsaw puzzle that most VWD diagnoses represent, and there are many other influences on, or modifiers of, plasma VWF level and function, other than the VWF gene (including the ABO blood group, epigenetic events, platelet and endothelial cell activity, hormonal influences [e.g., menstrual and pregnancy related], ADAMTS13 level and activity, and other factors related to the manufacture, storage, secretion, proteolysis and clearance of VWF that we have yet to identify [6]). Various additional environmental factors can also contribute to alter VWF levels, including stress, exercise, medications, illness, and inflammation. In total, then, there may be a large disconnection between the VWF genetic situation (i.e., mutations, polymorphisms) and the measured phenotype. Also, in most VWD investigations, the likelihood of successful genetic testing is low, likely to require an expensive and exhaustive evaluation of the entire VWF gene, and clinical utility low, Fig. 1. (A) Pre- and post-desmopressin (DDAVP) values for factor VIII (FVIII):coagulant (C) given the usual choices are similar and limited (DDAVP and/or and various VWF parameters. Dashed horizontal line indicates a nominal ‘normal’ VWF concentrate). cut-off value of 50 U/dL. VWD-1s = ‘severe’ type 1 von Willebrand disease (VWD) patient group (baseline von Willebrand factor [VWF] values <16 U/dl); VWD-1m = ‘moderate’ type 1 VWD patient group (baseline VWF values 16–35 U/dl); VWD-1p = ‘possible mild’ type 1 VWD patient group (baseline VWF values 36–65 U/dl); VWD-2A and VWD-2M = Types 2A VWD and 2M VWD patient groups, respectively. (B) Pre- and post-DDAVP ratios of collagen binding/antigen (CB/Ag) and ristocetin co-factor (RCo)/Ag for the same patient groups identified in (A). Dashed horizontal line indicates a nominal ‘normal’ cut-off value of 0.7 as discriminatory for functional VWF discordance (i.e., <0.7 is suggestive of discordance, and may reflect a type 2 VWD pattern). (C) Pre- and post-DDAVP PFA-100® closure-times (CTs) for the same patient groups identified in (A). Dashed horizontal line indicates the ‘normal’ cut-off value. Data represent a summary from references [23,24].
Acknowledgements The author would like to thank laboratory staff for performing the routine phenotypic testing of our VWD patients (Soma Mohammed, Jane McDonald, Ella Grezchnik). Conflict of Interest Statement The author has no conflict of interest related to the publication of this article.
E.J. Favaloro / Thrombosis Research 127 (2011) S17–S21
Funding None to declare. References [1] Sadler JE, Budde U, Eikenboom JC, Favaloro EJ, Hill FG, Holmberg L, et al.; Working Party on von Willebrand Disease Classification. Update on the pathophysiology and classification of von Willebrand disease: a report of the Subcommittee on von Willebrand Factor. J Thromb Haemost 2006;4:2103–14. [2] Favaloro EJ. Identification and functional characterisation of von Willebrand Disease. US Hematology 2009;2(1):16–23. [3] Favaloro EJ. Towards a new paradigm for the identification and functional characterisation of von Willebrand disease. Semin Thromb Hemost 2009;35: 60–75. [4] Favaloro EJ. An update on the von Willebrand factor collagen binding (VWF:CB) assay: 21 years of age and beyond adolescence, but not yet a mature adult. Semin Thromb Hemost 2007;33:727–44. [5] Favaloro EJ, Krigstein M, Koutts J, Brighton T, Lindeman R. Genetic testing for the diagnosis of von Willebrand Disease: benefits and limitations. J Coag Disorders 2010;2:37–47. [6] Favaloro EJ. Genetic testing for von Willebrand disease: the case against. J Thromb Haemost 2010;8:6–12. [7] Rodeghiero F, Tosetto A, Abshire T, Arnold DM, Coller B, James P, et al.; ISTH/SSC joint VWF and Perinatal/Pediatric Hemostasis Subcommittees Working Group. ISTH/SSC bleeding assessment tool: a standardized questionnaire and a proposal for a new bleeding score for inherited bleeding disorders. J Thromb Haemost 2010 Sep;8(9):2063–5. [8] Favaloro EJ, Lippi G, Franchini M. Contemporary platelet function testing. Clin Chem Lab Med 2010 May;48(5):579–98. [9] Othman M, Favaloro EJ. Genetics of type 2B von Willebrand disease: “true 2B,” “tricky 2B,” or “not 2B.” What are the modifiers of the phenotype? Semin Thromb Hemost 2008;34:520–31. [10] Federici AB, Mannucci PM, Castaman G, Baronciani L, Bucciarelli P, Canciani MT, et al. Clinical and molecular predictors of thrombocytopenia and risk of bleeding in patients with von Willebrand disease type 2B: a cohort study of 67 patients. Blood 2009;113(3):526–34. [11] Ribba AS, Loisel I, Lavergne JM, Juhan-Vague I, Obert B, Cherel G, et al. Ser968Thr mutation within the A3 domain of von Willebrand factor (VWF) in two related patients leads to a defective binding of VWF to collagen. Thromb Haemost 2001;86(3):848–54. [12] Riddell AF, Gomez K, Millar CM, Mellars G, Gill S, Brown SA, et al. Characterization of W1745C and S1783A: 2 novel mutations causing defective collagen binding in the A3 domain of von Willebrand factor. Blood 2009 Oct 15;114(16):3489–96. [13] Flood VH, Lederman CA, Wren JS, Christopherson PA, Friedman KD, Hoffmann RG, et al. Absent collagen binding in a VWF A3 domain mutant: utility of the VWF:CB in diagnosis of VWD. J Thromb Haemost 2010;8:1431–2.
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Thrombosis Research j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t h r o m r e s
Rethinking the diagnosis of von Willebrand disease Emmanuel J. Favaloro* Department of Haematology, Institute of Clinical Pathology and Medical Research (ICPMR), Westmead Hospital, NSW, Australia
article info
abstract
Keywords: von Willebrand disease VWD Diagnosis Desmopressin DDAVP Collagen binding Genetic testing
von Willebrand disease (VWD) is the most common inherited bleeding disorder and arises from deficiencies and/or defects in the plasma protein von Willebrand factor (VWF). VWD is classified into 6 different types, with type 1 identified as a (partial) quantitative deficiency of VWF, type 3 defined by a (virtual) total deficiency of VWF, and type 2 identifying four separate types (2A, 2B, 2M, 2N) characterised by qualitative defects. The classification is based on phenotypic assays including FVIII, VWF:Ag and VWF activity, typically by ristocetin cofactor (VWF:RCo), but also increasingly by collagen binding (VWF:CB). Phenotypic testing may be supplemented by multimer analysis, RIPA, and VWF:FVIII binding. Although genetic analysis is not required to diagnose VWD or to define a classification type, it may be useful in discrete situations. The current review briefly covers this diagnostic process, with a focus on newer approaches, including extended test panels and the use of data from desmopressin challenges as a diagnostic tool. © 2010 Elsevier Ltd. All rights reserved.
Abbreviations CB/Ag: DDAVP: FVIII: FVIII:C: GPIBA: HMW: LOD: RCo/Ag: RIPA: VWD: VWF: VWF:Ag: VWF:CB: VWF:FVIIIB: VWF:RCo:
collagen binding to antigen ratio desmopressin factor VIII factor VIII coagulant glycoprotein Iba high molecular weight limit of detection ristocetin cofactor to antigen ratio ristocetin-induced platelet agglutination(/aggregation) von Willebrand disease von Willebrand factor von Willebrand factor antigen von Willebrand factor collagen binding von Willebrand factor–Factor VIII binding von Willebrand factor ristocetin cofactor.
von Willebrand disease and von Willebrand factor von Willebrand disease (VWD) is the most common inherited bleeding disorder and arises from deficiencies and/or defects in the plasma protein von Willebrand factor (VWF) [1]. VWD is classified into 6 different types: type 1 (a partial quantitative deficiency of VWF), type 3 (a total deficiency of VWF), and types 2A, 2B, 2M, and 2N (characterising qualitative defects) [1] (Table 1). The * Correspondence: Dr. E. J. Favaloro, Department of Haematology, Institute of Clinical Pathology and Medical Research (ICPMR), Westmead Hospital, WSAHS, Westmead, NSW, 2145, Australia. Tel.: +61 2 9845 6618; fax: +61 2 9689 2331. E-mail address: [email protected] (E.J. Favaloro). 0049-3848 /$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
classification is based on phenotypic assays (factor VIIII [FVIII], VWF antigen [VWF:Ag] and VWF activity, usually by ristocetin cofactor [VWF:RCo] or collagen binding [VWF:CB]) [1–4]. Phenotypic testing may also be supplemented by VWF multimer analysis, ristocetin-induced platelet agglutination (RIPA), VWF:FVIII binding, and in select cases genetic analysis [1–6]. Diagnosis of VWD in an individual also requires establishing a personal history of bleeding, and this can be facilitated using a bleeding score [7]. Moreover, bleeding tendency in individuals with presumptive VWD may vary for reasons other than VWF [6]. In particular, the contribution of potential compound defects including platelet-related disorders is becoming increasing recognized [6]. Given similarities in bleeding symptoms, VWD and platelet function defects may also require application of a differential diagnostic process [8]. VWF has two main recognized functions to facilitate arrest of bleeding following injury – (i) promoting adhesion of platelets to each other and to the vasculature (‘primary hemostasis’), and (ii) binding and stabilizing FVIII (as used in ‘secondary’ hemostasis). However, VWF contains several functional binding sites in addition to those classically recognised as facilitating binding to its major platelet receptor glycoprotein Iba (GPIBA) and to FVIII, including binding sites for other platelet receptors (e.g., aIIbb3) and subendothelial matrix components such as collagen. The steadystate VWF plasma concentration and composition is also controlled by a complex process of manufacture, storage, secretion, proteolysis and clearance [1], with only some mechanisms elucidated. The heterogeneity of VWD therefore reflects the large and complex multimeric protein that VWF represents, as well as the simple fact that many factors additional to the VWF gene can influence the phenotypic presentation and an individual’s bleeding risk or clinical presentation [6].
Markedly decreased binding affinity for factor VIII
2N
Virtually complete deficiency of VWF
Decreased VWF-dependent platelet adhesion without a selective deficiency of HMW VWF multimers
2M
Rare form of VWD in developed countries (~1–5 cases per million population)
Rare form (<10%) of Type 2 VWD (~1–5 cases per million population).
Under-recognised form of Type 2 VWD.
Rare form (<10%) of Type 2 VWD (~1–5 cases per million population). Defined by enhanced responsiveness in a RIPA assay.
Most common presentation of Type 2 VWD.
Most common presentation of ‘VWD’ to laboratory, with most patients presenting with mildly reduced levels of VWF.
Comments
Typically defined by VWF levels <1% and FVIII <10%.
Defined by VWF:FVIIIB assay, with low FVIIIB/VWF ratios.
Low to normal levels of VWF, usually with VWF functional discordance detected by RCo/Ag generally <0.7, but relatively normal CB/Ag ratio. HMW VWF present, but multimers may show other abnormalities.
Low to normal levels of VWF, typically with VWF functional discordance (i.e., ratios of RCo/Ag and CB/Ag generally <0.7), loss of HMW VWF and (mild) thrombocytopenia. Atypical cases may not show this pattern.
Loss of HMW VWF. Usually low levels of VWF, with VWF functional discordance (i.e., ratios of RCo/Ag and CB/Ag typically <0.7).
Low levels of VWF, with VWF functional concordance (i.e., ratio of functional VWF/VWF:Ag approximates unity). RIPA usually normal unless VWF <20 U/dL.
Phenotypic presentation
Variable clinical response to DDAVP (some efficacy in some patients). Typically, for platelet-binding defect cases, VWF:Ag, VWF:CB & FVIII all rise (2–4× baseline), but VWF:RCo does not, with CB/Ag ratio remaining above 0.7 and RCo/Ag ratio remaining below 0.7.
Sometimes misidentified as type 1 or 2A VWD (e.g., LOD issues; assay variability, especially VWF:RCo; limited test panels).
Sometimes misidentified as (severe) type 1 VWD (e.g., LOD issues) or hemophilia A (if VWF testing not performed)
DDAVP ineffective.
Variable clinical response to DDAVP (some efficacy in some patients), depending on composite defect present.
DDAVP use contentious. DDAVP believed by some to be contraindicated, whereas others believe DDAVP represents an effective treatment in a proportion of patients. Effect of DDAVP on VWF and FVIII depends on defect. All parameters will rise initially, but may fall quickly depending on clearance mechanisms and defect.
Often misidentified as not VWD, or as type 1 VWD; sometimes misidentified as type 2A VWD (especially if VWF:CB or RIPA not performed, since VWF:Ag and/or RCo/Ag ratio sometimes normal)
Sometimes misidentified as hemophilia A/Carrier, or other forms of VWD (especially type 1)
Variable clinical response to DDAVP (some efficacy in some patients). Typically, VWF:Ag & FVIII rise (2–4× baseline), but VWF:RCo & VWF:CB do not, with CB/Ag and RCo/Ag ratios remaining below 0.7.
Usually respond well to DDAVP, unless VWF <10%. Generally, VWF:Ag, VWF:RCo, VWF:CB & FVIII all appreciably rise (2–4× baseline), with CB/Ag and RCo/Ag ratios remaining above 0.7.
Sometimes misidentified as type 2 VWD (e.g., LOD issues; assay variability, especially VWF:RCo; preanalytical issues) or misidentified as not VWD (e.g., acute phase increase in VWF due to stress, pregnancy)
Sometimes misidentified as type 1 or 2M VWD (e.g., LOD issues; assay variability, especially VWF:RCo, limited test panels)
Desmopressin (DDAVP) response profile
Phenotypic problems
Classification scheme derived and adapted from reference [1]. CB/Ag: collagen binding to antigen ratio; DDAVP: desmopressin; HMW: high molecular weight; FVIII:C: factor VIII coagulant; LOD: limit of detection; RCo/Ag: ristocetin cofactor to antigen ratio; RIPA: ristocetin induced platelet agglutination (/aggregation); VWD: von Willebrand disease; VWF: von Willebrand factor; VWF:CB: von Willebrand factor collagen binding; VWF:Ag: von Willebrand factor antigen; VWF:FVIIIB: VWF FVIII binding assay; VWF:RCo: von Willebrand factor ristocetin cofactor.
3
Increased affinity of VWF for platelet glycoprotein Ib
2B
Qualitative VWF defects
2
Decreased VWF-dependent platelet adhesion and a selective deficiency of high-molecular-weight VWF multimers
Partial quantitative deficiency of VWF
1
2A
Description
VWD type
Table 1 Classification scheme for von Willebrand disease, phenotypic presentation, phenotypic problems and desmopressin response profile
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Contribution of phenotypic characterisation to diagnosis of VWD
Contribution of phenotypic characterisation to misdiagnosis of VWD
In type 1 VWD there is a partial deficiency of VWF with similar (concordant) reductions in VWF:Ag, FVIII:C, VWF:RCo and VWF:CB. If performed, multimeric analysis would show a normal multimeric structure and RIPA would usually be normal unless levels of VWF fall below 10–20 U/dL. In type 3 VWD, VWF is (virtually) absent and all VWF tests would (in theory) yield values around 0 U/dL. If performed, multimeric analysis would show a lack of VWF and RIPA would be absent (no response to ristocetin). In type 2 VWD, VWF:Ag is usually low, but may be normal; however, functional tests will show an abnormality or reduction, with the pattern helping to identify the type [1–4]; summarised as follows: (i) In type 2A VWD, the defect is caused by a loss of high molecular weight (HMW) VWF, due either to faulty assembly or enhanced proteolysis [1]. This deficiency can be identified using multimer analysis, or by assessing VWF activity, with values of VWF:RCo and VWF:CB both lower than VWF:Ag. This test pattern is termed VWF functional discordance, and expressed numerically using ratios. Type 2A VWD is therefore typically characterised by VWF:RCo/VWF:Ag (RCo/Ag) and VWF:CB/VWF:Ag (CB/Ag) ratios below around 0.7. (ii) Type 2B VWD is characterised by enhanced binding (i.e., hyper-adhesive activity) of VWF to GPIBA, as identified by elevated RIPA responsiveness. This enhanced VWF–GPIBA binding typically causes some clearance or loss of HMW VWF (the most adhesive forms) and also VWF-bound platelets (thus mild thrombocytopenia). However, some ‘atypical’ forms of 2B VWD may not show these latter features [9,10]. Like 2A VWD, HMW VWF loss can be identified using multimer analysis, or surrogate markers such as VWF:CB and VWF:RCo. Phenotypically, then, type 2B VWD cases (like 2A) typically express RCo/Ag and CB/Ag ratios below around 0.7, particularly under periods of stress, and this pattern may lead to some 2B VWD patients being misidentified as 2A where RIPA is not performed. (iii) Type 2M VWD is recognised as an inherent VWF defect, where loss of VWF function is not due to loss of HMW VWF. Thus, although multimer analysis may show some structural abnormalities, HMW multimers will be present, and the pattern may more closely resemble type 1 VWD. Most cases of type 2M have defective binding to GPIBA; hence, VWF:RCo, which ‘functionally’ detects this binding, tends to be lower than VWF:Ag, and RCo/Ag ratios are below around 0.7. However, since collagen binding is less affected, VWF:CB values are similar to VWF:Ag (i.e., show concordance), and CB/Ag ratios are typically above 0.7. Interestingly, rare forms of 2M VWD show the opposite pattern, since the VWF defect affects collagen binding but not GPIBA binding; phenotypically this leads to low CB/Ag ratios, but normal RCo/Ag ratios [1–4,11– 13]. (iv) Type 2N VWD represents an inherent VWF defect causing defective FVIII binding. Thus, plasma FVIII is labile, prone to proteolysis, and plasma FVIII:C levels tend to be lower than VWF (i.e., low plasma FVIII/VWF ratios are evident). Phenotypically, 2N VWD tends to mimic hemophilia A or hemophilia A carriers, but can be identified using the VWF:FVIIIB assay, with low FVIIIB/VWF ratios in 2N VWD, but normal ratios in Hemophilia A (/carriers) [14].
Several major problems with phenotypic characterisation of VWD lead to ineffective diagnosis in some cases, and to misdiagnosis in others. First, there is a continuum of VWF values in all normal individuals, as well as most individuals with VWD, leading to overlaps in values between unaffected ‘normals’ and affected VWD individuals [15]. Indeed, the usual methods for establishing reference ranges for phenotype based assays (i.e. mean +/− 2 × standard deviation) by definition ascribes ~2.5% of the normal population as having a low level of VWF. Second, VWF is an acutephase reactant, and increases in times of stress, illness, infection, as well as during pregnancy. Third, many of the assays used, particularly VWF:RCo, show a high degree of assay variability, meaning different test results on different occasions in the same or different laboratories [16]. Fourth, lower limit of detection (LOD) issues are evident for many VWF assays, again in particular VWF:RCo (which may be as high as 20%) or VWF when measured by latex immuno assay (LIA) technology [17]. This poses difficulty in VWD diagnostics in particular with the most severe forms of VWD. Fifth, preanalytical issues in VWD testing can lead to identification of VWD-like patterns in normal individuals, or to incorrect VWD types being assigned [18]. Sixth, most laboratories use inappropriate or limited test panels, insufficient to properly identify phenotypically all cases of VWD. Taking these factors into consideration, and depending on presiding test panels employed and test results obtained, patients with a mild form of ‘type 1 VWD’ could easily be misidentified as not having VWD (false negative), normal individuals may be misdiagnosed as having VWD (false positive), type 3 VWD individuals may be misdiagnosed as severe type 1 or type 2 VWD, and type 1 VWD might be misdiagnosed as type 2 VWD or vice versa [19,20]. For example, LOD issues lead to poor discrimination of type 3, severe type 1 and severe type 2 VWD, either because they cannot be differentiated, or because of false concordance/ discordance by RCo/Ag. High assay variability, particularly for VWF:RCo, also means that discordance by RCo/Ag will occasionally not be observed in types 2A and 2B VWD (leading to misdiagnosis as type 1), or a false functional discordance may instead be identified (leading to misdiagnosis of type 1 as type 2A). Preanalytical issues usually cause normal individuals or type 1 VWD individuals to be misidentified as type 2 [18]. Although performance of VWF multimers might assist the diagnosis of VWD, and ‘prevent’ some of these misdiagnoses, the reality of broadly applied multimer testing is similarly not ideal. Up to 20% and 40% of laboratories will respectively identify loss of HMW VWF in normal or type 1 VWD samples (i.e., false type 2 VWD) [21]. Even ‘expert’ centres using multimer analysis also misdiagnose VWD, as around 20% of cases identified with type 1 VWD are in fact type 2 VWD, with most of these being 2M VWD [22]. This latter finding is explained by false VWF functional concordance identified due to high assay variability and LOD issues with VWF:RCo, with multimer analysis failing to resolve between type 1 and type 2M VWD. Additional useful investigative approaches to help overcome phenotypic assay limitations Diagnostic problems from phenotypic evaluation can be minimised by: (i) using improved methodologies (e.g., enzyme linked immunosorbant assay [ELISA] in place of electro-immuno diffusion [Laurel gel]), (ii) extending the test panel, (iii) repeat testing using a fresh sample for confirmation, (iv) avoiding testing of stressed, ill or pregnant patients, and (v) using data from a desmopressin (DDAVP) challenge test.
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The addition of VWF:CB to a core test panel of VWF:RCo, VWF:Ag and FVIII:C, for example, will reduce phenotypic-based diagnostic error rates by around half [19,20]. DDAVP is a non-tranfusional form of therapy given to select patients with VWD that results in release of endogenous (endothelial-cell stored) VWF. Notably, DDAVP is (nearly invariably) effective in type 1 VWD where VWF levels are above 30 IU/dL, is (usually) effective in other cases of type 1, and is sometimes effective in type 2 VWD [25]. A trial of DDAVP is commonly performed to assess clinical utility, since responsiveness is typically stable over time within individuals, despite varying between individuals. There is additional but under-recognised utility in assessing data from DDAVP trials for the diagnosis of VWD, given the response profile is often VWD-type characteristic [2,3,23]. In brief, FVIII and all VWF test parameters rise post DDAVP in type 1, and RCo/Ag and CB/Ag ratios remain above 0.7 (Fig. 1). In type 2A, FVIII and VWF:Ag rise, but VWF:RCo and VWF:CB do not, or their response is transient; therefore RCo/Ag and CB/Ag ratios typically remain below 0.7. In GPIBA-binding dysfunction type 2M, FVIII, VWF:Ag and VWF:CB typically all rise; however, VWF:RCo does not – therefore CB/Ag ratios typically remain above 0.7 but RCo/Ag ratios remain below 0.7. Thus, in patients where the VWD type is unclear, DDAVP response patterns can help assign the VWD type. The PFA-100 also has potential utility in this setting, as initially prolonged closure times will tend to shorten and normalise post DDAVP in type 1, but not in types 2A and 2M (Fig. 1; [2,3,24]). Finally, genetic testing in VWD may be of use in select investigations, namely in (i) type 2N (to help discrimination from hemophilia A/carrier); (ii) type 2B VWD (primarily for discrimination from platelet-type [PT]-VWD); (iii) type 3 VWD (for prenatal assessment/family studies and alloantibody risk assessment); and perhaps in (iv) type 2A/2M VWD and (v) type 1 VWD where VWF levels are <20–30 IU/mL [5]. In these cases, testing is focused and there may be therapeutic implications to an incorrect diagnosis (e.g., VWF concentrate vs FVIII concentrate in 2N VWD vs hemophilia; VWF concentrate vs platelet replacement in 2B VWD vs PT-VWD). However, genetic testing cannot be broadly applied to VWD diagnostics, as it typically provides only one piece of the jigsaw puzzle that most VWD diagnoses represent, and there are many other influences on, or modifiers of, plasma VWF level and function, other than the VWF gene (including the ABO blood group, epigenetic events, platelet and endothelial cell activity, hormonal influences [e.g., menstrual and pregnancy related], ADAMTS13 level and activity, and other factors related to the manufacture, storage, secretion, proteolysis and clearance of VWF that we have yet to identify [6]). Various additional environmental factors can also contribute to alter VWF levels, including stress, exercise, medications, illness, and inflammation. In total, then, there may be a large disconnection between the VWF genetic situation (i.e., mutations, polymorphisms) and the measured phenotype. Also, in most VWD investigations, the likelihood of successful genetic testing is low, likely to require an expensive and exhaustive evaluation of the entire VWF gene, and clinical utility low, Fig. 1. (A) Pre- and post-desmopressin (DDAVP) values for factor VIII (FVIII):coagulant (C) given the usual choices are similar and limited (DDAVP and/or and various VWF parameters. Dashed horizontal line indicates a nominal ‘normal’ VWF concentrate). cut-off value of 50 U/dL. VWD-1s = ‘severe’ type 1 von Willebrand disease (VWD) patient group (baseline von Willebrand factor [VWF] values <16 U/dl); VWD-1m = ‘moderate’ type 1 VWD patient group (baseline VWF values 16–35 U/dl); VWD-1p = ‘possible mild’ type 1 VWD patient group (baseline VWF values 36–65 U/dl); VWD-2A and VWD-2M = Types 2A VWD and 2M VWD patient groups, respectively. (B) Pre- and post-DDAVP ratios of collagen binding/antigen (CB/Ag) and ristocetin co-factor (RCo)/Ag for the same patient groups identified in (A). Dashed horizontal line indicates a nominal ‘normal’ cut-off value of 0.7 as discriminatory for functional VWF discordance (i.e., <0.7 is suggestive of discordance, and may reflect a type 2 VWD pattern). (C) Pre- and post-DDAVP PFA-100® closure-times (CTs) for the same patient groups identified in (A). Dashed horizontal line indicates the ‘normal’ cut-off value. Data represent a summary from references [23,24].
Acknowledgements The author would like to thank laboratory staff for performing the routine phenotypic testing of our VWD patients (Soma Mohammed, Jane McDonald, Ella Grezchnik). Conflict of Interest Statement The author has no conflict of interest related to the publication of this article.
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