Haemophilia (2014), 20 (Suppl. 4), 59–64
DOI: 10.1111/hae.12414
REVIEW ARTICLE
von Willebrand disease and platelet disorders † S . J . I S R A E L S ‡ and S . A . B R O W N § ¶ E . J . F A V A L O R O , * I . B O D O, *Diagnostic Haemostasis, Haematology Department Institute of Clinical Pathology and Medical Research (ICPMR), Pathology West, Westmead Hospital, Westmead, NSW, Australia; †Department of Hematology and Stem Cell Transplantation, St. Istv an & St. L aszl o Hospital, Budapest, Hungary; ‡Department of Pediatrics and Child Health, University of Manitoba, Winnipeg, MB, Canada; §Department of Haematology & Haemophilia Centre, Royal Children’s Hospital; and ¶Department of Haematology, Pathology Queensland, Brisbane, Qld, Australia
Summary. The diagnosis and management of bleeding disorders is made difficult by the complexity and variety of disorders, clinical symptoms and bleeding type and severity. von Willebrand disease (VWD) and platelet disorders are disorders of primary haemostasis and together represent the most common inherited
bleeding disorders. In this article, we describe the diagnosis of VWD and platelet disorders and the treatment options for VWD.
Introduction
recognizes six subtypes of VWD [3]. Type 1 represents a partial quantitative deficiency of a functionally normal VWF protein. Type 3 VWD represents a severe (complete) deficiency of VWF. Type 2 VWD represents a group of qualitative VWF defects that comprise (i) type 2A VWD [loss of high molecular weight (HMW) VWF], type 2B VWD (enhanced functional binding of VWF to platelets that typically leads to loss of HMW VWF and mild thrombocytopenia), (iii) 2N VWD (loss of VWF-FVIII binding) and (iv) 2M VWF (VWF dysfunction not associated with loss of HMW VWF). The proper identification of VWD and its type is important as it has therapeutic implications [4]. In practice, VWD and its type can be determined by a process of laboratory testing that encompasses a comprehensive panel of different tests [1,5,6] (Table 1). The two main tests employed by virtually all laboratories are VWF antigen (VWF:Ag) and FVIII coagulant (FVIII:C); these, respectively, measure the level of VWF protein and FVIII activity. The most common VWF activity based test is the ristocetin cofactor (VWF:RCo) assay, which essentially measures VWF binding to the platelet VWF receptor GPIb. An additional test used by a proportion of laboratories is the collagen binding (VWF:CB) assay; collagen is a sub-endothelial matrix component which binds VWF in vivo. Additional ‘activity’ assays in general practice include those more recently released from commercial suppliers, such as the Werfen-IL and Siemens Innovance activity assays. VWF can also be assessed by other methods including multimer analysis to assess for loss of HMW VWF as well as structural abnormalities.
The diagnosis and management of von Willebrand disease (VWD) remains problematic for many laboratories and clinicians [1]. VWD arises from deficiency and/or defects of von Willebrand factor (VWF), a multimeric adhesive plasma protein essential for effective primary haemostasis. VWF is a multifunctional protein [2], which explains the heterogeneity in clinical symptoms and bleeding risk, as well as diagnostic challenges. Inherited platelet disorders include abnormalities of both number and function. Our understanding of specific rare platelet disorders has improved significantly in the last decade with the identification of specific disease-causing mutations. However, the investigation of individual patients with mild/moderate platelet disorders remains a challenge, as diagnostic tools available in most clinical laboratories often do not provide a definitive diagnosis. Improving our ability to define the abnormalities in common platelet disorders is our next challenge.
Challenges in the laboratory diagnosis of VWD The most recent classification scheme from the International Society on Thrombosis and Haemostasis Correspondence: Simon A. Brown, Department of Haematology, Pathology Queensland, Level 3 Block 7, Royal Brisbane & Women’s Hospitals, Herston, Brisbane, Qld 4029, Australia. Tel.: +617 3636 9846; fax: +617 3636 1552; e-mail: [email protected] Accepted 24 February 2014 © 2014 John Wiley & Sons Ltd
Keywords: desmopressin, platelet aggregation, platelet disorders, von Willebrand disease, von Willebrand factor
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Table 1. A practical guide to identification of von Willebrand disease and it’s type. VWD type
VWF:Ag
VWF:RCo
VWF:CB
FVIII
Multimers
1
↓ to ↓↓
↓ to ↓↓
↓ to ↓↓
N to ↓↓
2A
↓ to ↓↓
↓↓ to ↓↓↓
↓↓ to ↓↓↓
↓ to ↓↓
2B
N to ↓↓
↓ to ↓↓↓
↓ to ↓↓↓
N to ↓↓
2N
N to ↓↓
N to ↓↓
N to ↓↓
↓ to ↓↓↓
2M
↓ to ↓↓
↓ to ↓↓↓
↓ to ↓↓↓
↓ to ↓↓
No loss of HMW VWF; some multimer defects may however be observed
3
↓↓↓ (absent)
↓↓↓ (absent)
↓↓↓ (absent)
↓↓↓
No VWF present
Normal pattern but reduced intensity Loss of HMW VWF Loss of HMW VWF Normal pattern
RCo/Ag
CB/Ag
FVIII/Ag
>0.7
>0.7
>0.7
0.7
0.7
NA
Comments
2A and 2B VWD can only be distinguished by means of RIPA Similar to haemophilia A pattern; distinguish using VWF:FVIII binding assay or genetic analysis 2A and 2M VWD can only be distinguished by composite multipanel VWF testing Type 3 VWD can only be identified when VWF tests are sensitive to very low levels
Ag, antigen; CB, collagen binding; FVIII, factor VIIII; N, normal; NA, not applicable; RCo, ristocetin cofactor; RIPA, ristocetin induced platelet aggregation; VWF, von Willebrand factor.
In brief, type 1 VWD can be identified as a deficiency of VWF, with the level of deficiency correlating with the severity of the disorder. In these cases, low levels of VWF:Ag, VWF:RCo, VWF:CB and other activity assays (‘VWF:Act’) will be determined by laboratories testing patient plasma. However, as the VWF is functionally normal, similar (‘concordant’) levels of VWF will be identified using all VWF assays, and the ratio of any VWF assay to another will be close to one. In practice, a low level of VWF together with a ratio of VWF activity (VWF:RCo, VWF:CB or VWF:Act) to VWF:Ag above 0.7 is consistent with type 1 VWD (Table 1). In contrast, in type 2 VWD, VWF activity based assays will identify some VWF defect, with the defect identified helping to characterize the VWD type. Thus, loss of HMW VWF (present in 2A and 2B VWD) can be identified directly by multimer analysis or indirectly by a relatively larger reduction in VWF:RCo, VWF:CB and VWF:Act compared to VWF:Ag. This ‘VWF discordance’ can be expressed by a ratio of VWF activity to VWF:Ag below 0.5–0.7. Type 2M reflects a variety of functional defects, with most representing a platelet GP-Ib binding defect; hence VWF:RCo/VWF:Ag will usually be low, but VWF:CB/VWF:Ag may be normal. Type 2N VWD reflects a loss of VWF-FVIII binding; hence FVIII/VWF:Ag will be low, and phenotypically these patients resemble mild haemophilia A. The main problems relating to laboratory identification of VWD and its type are high inter-laboratory
Haemophilia (2014), 20 (Suppl. 4), 59--64
and inter-method assay variability, problems with lower limit of VWF detection, performance of insufficient test panels by laboratories to appropriately define all forms of VWD, and challenges in the interpretation of test findings. Thus, most laboratories struggle with differentiation between severe type 1 vs. 3 VWD, type 2M vs. 2A, 2M vs. 1, 2A vs. 2B, 2N vs. haemophilia A and type 3 VWD vs. haemophilia A. Severe type 1 and 3 VWD can only be distinguished if laboratories perform assays that are capable of detecting VWF levels down to 50 U dL 1 (>80–100 for critical bleeding or high-risk major surgery) for 7–14 days in case of major surgery and 1–5 days for minor procedures. A typical loading dose to achieve this is 50 VWF:RCo U kg 1 followed by 20–40 U kg 1 maintenance dose every 8–12 h for major surgery and 12–18 h for minor procedures. Daily VWF:RCo and FVIII determinations are mandatory to keep adequate levels of VWF and avoid ultra-high levels (i.e. >200 IU dL 1) of FVIII [9,20]. Standard thromboprophylaxis should be used in patients in whom VWF levels are normalized.
Management of VWD In general, three main modalities are available in our therapeutic armamentarium to stop bleeding in VWD patients: correction of the VWF plasma levels by (i) DDAVP or (ii) factor replacement, and (iii) decreasing the bleeding tendency by manipulating alternative routes with antifibrinolytic agents or oestrogen–progesterone drugs. These three approaches are not mutually exclusive.
Desmopressin (DDAVP) Desmopressin (1-deamino-8-d-arginine vasopressin) is a synthetic analogue of vasopressin [8,9]. DDAVP [Emosintâ (Kedrion, Pascoti Barga, Italia), Minirinâ (Ferring AB, Malmo, Sweden), Octostimâ (Ferring AB, Malmo, Sweden)] is inexpensive and devoid of any risk of transmitting blood-borne infections. It should be the preferred treatment modality, whenever possible. Usual administration is via the intravenous route, in a dosage of 0.3 lg kg 1, as an infusion over 20–30 min. A test infusion is recommended at the time of diagnosis to predict future response [10,11]. Desmopressin infusions are usually well tolerated, with tachycardia, headaches and flushing being the main side effects, usually ameliorated by slowing the infusion. Repeated DDAVP doses often become ineffective (a phenomenon called tachyphylaxis). This
Long-term secondary prophylaxis. Over the past decade, it has become clear that in severe forms of VWD, long-term prophylaxis is beneficial [21–23].
Adjunctive treatments by manipulating alternative routes of haemostasis Antifibrinolytic agents. As mucosal surfaces are rich in fibrinolytic activity [9], blocking fibrinolysis is a useful adjunctive measure to stop bleeding. Epsilon-aminocaproic acid (at a dose of 50–60 mg kg 1 every 4– 6 h) or tranexamic acid (at a dose of 10–15 mg kg 1
Table 2. Selected plasma-derived factor concentrates typically used for the treatment of VWD*. Product
Alphanate
Biostate
Fandhi
Manufacturer Purification
Grifols Heparin CTG
CSL Behring Precipitation + heparin CTG
Grifols Heparin CTG
Viral inactivation
S/D + dry heat (80°C, 72 h)
S/D + dry heat (80°C, 72 h)
FVIII IU mg 1 VWF:RCo/Ag VWF:RCo/FVIII
>100
50 0.94 1.21
0.8 2.0
Haemate P‡
Wilate
Wilfactin
Octapharma Ion ex. + size exclusion
LFB Ion ex. + affinity
S/D + dry heat (80°C, 72 h)
CSL Behring Multiple electrolyte precipitation Pasteurization (60°C, 10 h)
S/D + dry heat (100°C, 2 h)
>100
40
>80
S/D, 35 nm NF, dry heat (80°C, 72 h) >50† 0.95 >10
0.83 1.48
0.96 2.54
0.7 0.8–1.0
CTG, chromatography; Ion ex., ion exchange chromatography; S/D, solvent/detergent treatment; NF, nanofiltration. *Data from references no. 18 and 19. † Given as VWF:RCo/mg. ‡ Humate P in the US.
© 2014 John Wiley & Sons Ltd
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every 8–12 h) may be administered orally, intravenously, or topically [9]. Oestrogen–progesteron preparations render the endometrium less susceptible to bleeding, and may be very useful in managing menorrhagia in VWD patients [8,9].
make it possible to use them as a screen prior to laboratory testing. However, existing tools have low specificity and will not provide a definitive diagnosis [27,28].
Investigation of platelet function abnormalities
Diagnostic approach to platelet disorders As there are no population-based data, the prevalence of inherited platelet disorders, which encompass both functional disorders and thrombocytopenia (Table 3), remains unknown. In studies of patients presenting with mucocutaneous bleeding, platelet abnormalities are at least as common as VWD. Severe disorders are often recognized in childhood, but mild disorders may go undiagnosed unless there is a family history that prompts testing, or until a haemostatic challenge results in significant bleeding. Algorithms have been developed to aid with the investigation of inherited platelet function disorders [24] (available at: www.ahcdc.ca/index.php/research/rare-inherited-bleeding-disorders) [25], and thrombocytopenias [26]. Validated bleeding assessment tools (BATs) are useful in standardizing information obtained from the patient history and accurately recording the severity and frequency of bleeding symptoms [27]. The high negative predictive value of some of these tools may
There is no ideal simple, inexpensive, sensitive screening test that reliably identifies patients requiring specialized testing of platelet function. Although both bleeding times and PFA-100/200â closure times have been used for this purpose, these tests are not adequately sensitive to rule out the need for further testing [29], and should be considered optional. A validated BAT may be more useful in assessing a patient’s bleeding propensity and determining whether further specialized laboratory investigations are warranted. The most widely used method for assessing platelet function is light transmission aggregometry (LTA), in which the change in optical density of a stirred sample of citrated platelet-rich plasma is measured by a photometer following the addition of agonists. Although many pre-analytical and analytical variables affect the results, and international surveys have shown that there is wide variation in methodology, LTA remains the gold standard platelet function test. Recommenda-
Table 3. Inherited platelet disorders. Inherited disorders of platelet function Abnormalities of receptors for adhesive proteins GPIb-IX-V complex (Bernard–Soulier syndrome*, platelet-type VWD*) GPIIb-IIIa (aIIbb3) (Glanzmann thrombasthenia) GPIa-IIa (a2b1) GPVI GPIV Abnormalities of receptors for soluble agonists Thromboxane A2 receptor P2Y12 receptor a2-adrenergic receptor Abnormalities of platelet granules d-granules (d-storage pool deficiency, Hermansky–Pudlak, Chediak–Higashi, TAR syndrome*) a-granules (Gray platelet syndrome*, ARC syndrome*, Quebec platelet disorder*, Paris–Trousseau–Jacobsen syndrome*) a- and d-granules (a,d-storage pool deficiency) Abnormalities of signal-transduction pathways Primary secretion defects Abnormalities of the arachidonic acid/thromboxane A2 pathway Gaq deficiency Partial selective PLC-b2 deficiency Defects in pleckstrin phosphorylation Defects in Ca2+ mobilization Abnormalities of cytoskeleton MYH9-related disorders (May–Hegglin anomaly, Sebastian syndrome, Fechtner syndrome, Epstein syndrome)* Wiskott–Aldrich syndrome* X-linked thrombocytopenia* Abnormalities of membrane phospholipids Scott syndrome*
Inherited thrombocytopenias Small platelets Wiskott-Aldrich syndrome† X-linked thrombocytopenia Normal-sized platelets Congenital amegakaryocytic thrombocytopenia Amegakaryocytic thrombocytopenia with radio-ulnar synostosis Thrombocytopenia with absent radii† Familial platelet disorder and predisposition to acute myeloid leukemia† Autosomal dominant thrombocytopenia Large platelets Bernard-Soulier syndrome† Velocardiofacial (22q deletion) syndrome Platelet-type von Willebrand disease† von Willebrand Disease Type 2B Gray platelet syndrome† ARC syndrome MYH9-related disorders† X-linked thrombocytopenia with thalassemia Paris–Trousseau–Jacobsen syndrome Dyserythropoietic anemia with thrombocytopenia
*Usually present with thrombocytopenia in addition to functional abnormalities. † May present with functional abnormalities in addition to thrombocytopenia.
Haemophilia (2014), 20 (Suppl. 4), 59--64
© 2014 John Wiley & Sons Ltd
VWD AND PLATELET DISORDERS
tions for standardization have recently been published [30]. Whole blood aggregometry that measures aggregation as the change in electrical impedance between two electrodes, as platelets adhere and aggregate in response to agonists, has the advantages of using smaller blood volumes than LTA and requiring less manipulation of the sample. Lumi-aggregometry, in which the secretion of ATP from the dense granules is measured by the use of a luciferin/luciferase reagent, is useful in the identification of secretion defects. The specific cause of secretion defects remains unknown in most patients, but quantitation of platelet dense granules by whole mount electron microscopy will identify a subgroup with dense granule deficiency. Additional laboratory investigations including flow cytometry and mutation analysis can provide specific diagnoses in patients with defined abnormalities [29,31].
Investigation of thrombocytopenia Investigation of thrombocytopenia can be guided by platelet size (Table 3) [24,26,32]. Classification into small, normal-sized, or large platelets on the basis of mean platelet volume (MPV) should be confirmed by evaluation of the blood film. Automated cell counters underestimate platelet counts when platelet size is outside of the established reference range, and therefore should be combined with the evaluation of the peripheral blood film to provide additional information about platelet number, size, clumping and granularity and morphology of leucocytes and red cells [33]. Evaluation of the patient and family for evidence of clinical features in addition to the thrombocytopenia may identify a syndromic aetiology. Some inherited thrombocytopenias are also associated with platelet dysfunction (Table 3). Importantly, the genetic bases for more inherited thrombocytopenias have been identified in the last decade, allowing confirmation of the diagnosis in the patient and family members [32,34].
References 1 Favaloro EJ. Von Willebrand disease: local diagnosis and management of a globally distributed bleeding disorder. Semin Thromb Hemost 2011; 37: 440–55. 2 Yee A, Kretz CA. von Willebrand factor: form for Function. Semin Thromb Hemost 2014; 40: 17–27. 3 Sadler JE, Budde U, Eikenboom JCJ et al. 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. 4 Favaloro EJ, Franchini M, Lippi G. Biological therapies for von Willebrand disease. Expert Opin Biol Ther 2012; 12: 551–64.
© 2014 John Wiley & Sons Ltd
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Phenotype/genotype correlations in mild platelet disorders Most individuals with platelet function disorders have abnormalities that are not clearly defined by standard clinical laboratory investigations. Some patients with mild/moderate bleeding and non-specific abnormalities of LTA have granule secretion defects [35], or subtle changes in receptor-mediated signal transduction. The investigation of these less well-defined abnormalities requires specialized testing, which may be beyond the capacity of most clinical laboratories. However, the definition of an individual family phenotype that directs sequencing of specific candidate genes has successfully identified previously unknown defects [36]. These are often heterozygous mutations that may represent only one of several abnormalities contributing to the bleeding phenotype; mild bleeding is likely to be a complex trait. The identification of subtle abnormalities as disease causing is complicated further by the fact that normal platelet reactivity is highly variable. Both hypo- and hyper-responsiveness to specific agonists have been shown to be associated with polymorphisms in genes encoding platelet adhesive protein receptors including integrins aIIbb3 and a2b1, and GPIb-IX-V [37]. More recently, genome-wide association studies have successfully identified proteins (some not previously suspected to be involved in platelet production or function) associated with variations in platelet count, MPV, and aggregation responses [38]. Study results vary depending on the phenotypic parameters chosen and the study population. However, these powerful tools are likely to change both our understanding of inherited platelet disorders and eventually how we investigate our patients with mucocutaneous bleeding.
Disclosures The authors stated that they had no interests which might be perceived as posing a conflict or bias.
5 Favaloro EJ. Diagnosis and classification of von Willebrand disease: a review of the differential utility of various functional von Willebrand factor assays. Blood Coagul Fibrinolysis 2011; 22: 553–64. 6 Favaloro EJ, Bonar R, Favaloro J, Koutts J. Diagnosis and management of von Willebrand disease in Australia. Semin Thromb Hemost 2011; 37: 542–54. 7 Favaloro EJ. Detailed von Willebrand factor multimer analysis in patients with von Willebrand disease in the European study, molecular and clinical markers for the diagnosis and management of type 1 von Willebrand disease (MCMDM-1VWD): a rebuttal. J Thromb Haemost 2008; 6: 1999–2001; author reply 2002-3.
8 Rodeghiero F, Castaman G, Tosetto A. How I treat von Willebrand disease. Blood 2009; 114: 1158–65. 9 Mannucci PM. Treatment of von Willebrand’s disease. N Engl J Med 2004; 351: 683–94. 10 Federici AB, Mazurier C, Berntorp E et al. Biologic response to desmopressin in patients with severe type 1 and type 2 von Willebrand disease: results of a multicenter European study. Blood 2004; 103: 2032–8. 11 Castaman G, Lethagen S, Federici AB et al. Response to desmopressin is influenced by the genotype and phenotype in type 1 von Willebrand disease (VWD): results from the European Study MCMDM-1VWD. Blood 2008; 111: 3531–9.
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12 Mannucci PM, Bettega D, Cattaneo M. Patterns of development of tachyphylaxis in patients with haemophilia and von Willebrand disease after repeated doses of desmopressin (DDAVP). Br J Haematol 1992; 82: 87–93. 13 Smith TJ, Gill JC, Ambruso DR, Hathaway WE. Hyponatremia and seizures in young children given DDAVP. Am J Hematol 1989; 31: 199–202. 14 Bond L, Bevan D. Myocardial infarction in a patient with hemophilia treated with DDAVP. N Engl J Med 1988; 318: 121. 15 Byrnes JJ, Larcada A, Moake JL. Thrombosis following desmopressin for uremic bleeding. Am J Hematol 1988; 28: 63–5. 16 Holmberg L, Nilsson IM, Borge L, Gunnarsson M, Sj€ orin E. Platelet aggregation induced by 1-desamino-8-D-arginine vasopressin (DDAVP) in Type IIB von Willebrand’s disease. N Engl J Med 1983; 309: 816–21. 17 Casonato A, Steffan A, Pontara E et al. PostDDAVP thrombocytopenia in type 2B von Willebrand disease is not associated with platelet consumption: failure to demonstrate glycocalicin increase or platelet activation. Thromb Haemost 1999; 81: 224–8. 18 Federici AB. The safety of plasma-derived von Willebrand/factor VIII concentrates in the management of inherited von Willebrand disease. Expert Opin Drug Saf 2009; 8: 203–10. 19 Castaman G. Treatment of von Willebrand disease with FVIII/VWF concentrates. Blood Transfus 2011; 9(Suppl. 2): s9–13. 20 Mannucci PM. How I treat patients with von Willebrand disease. Blood 2001; 97: 1915–9.
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21 Berntorp E, Petrini P. Long-term prophylaxis in von Willebrand disease. Blood Coagul Fibrinolysis 2005; 16(Suppl. 1): S23–6. 22 Federici AB. Highly purified VWF/FVIII concentrates in the treatment and prophylaxis of von Willebrand disease: the PRO. WILL Study. Haemophilia 2007; 13(Suppl. 5): 15–24. 23 Abshire TC, Federici AB, Alvarez MT et al. Prophylaxis in severe forms of von Willebrand’s disease: results from the von Willebrand Disease Prophylaxis Network (VWD PN). Haemophilia 2013; 19: 76–81. 24 Israels SJ, Kahr WH, Blanchette VS, Luban NL, Rivard GE, Rand ML. Platelet disorders in children: a diagnostic approach. Pediatr Blood Cancer 2011; 56: 975–83. 25 Favaloro EJ, Lippi G, Franchini M. Contemporary platelet function testing. Clin Chem Lab Med 2010; 48: 579–98. 26 Balduini CL, Cattaneo M, Fabris F et al. Inherited thrombocytopenias: a proposed diagnostic algorithm from the Italian Gruppo di Studio delle Piastrine. Haematologica 2003; 88: 582–92. 27 Rydz N, James PD. The evolution and value of bleeding assessment tools. J Thromb Haemost 2012; 10: 2223–9. 28 Lowe GC, Lordkipanidze M, Watson SP. Utility of the ISTH bleeding assessment tool in predicting platelet defects in participants with suspected inherited platelet function disorders. J Thromb Haemost 2013; 11: 1663–8. 29 Harrison P, Lordkipanidze M. Testing platelet function. Hematol Oncol Clin North Am 2013; 27: 411–41. 30 Cattaneo M, Hayward CP, Moffat KA, Pugliano MT, Liu Y, Michelson AD. Recom-
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mendations for the Standardization of Light Transmission Aggregometry: a Consensus of the WORKING party from the Platelet Physiology Subcommittee of SSC/ISTH. J Thromb Haemost 2013; 11: 1183–9. Nurden AT, Nurden P. Congenital platelet disorders and understanding of platelet function. Br J Haematol 2013; doi: 10. 1111/bjh.12662. Geddis AE. Inherited thrombocytopenias: an approach to diagnosis and management. Int J Lab Hematol 2013; 35: 14–25. Latger-Cannard V, Fenneteau O, Salignac S, Lecompte TP, Schlegel N. Platelet morphology analysis. Methods Mol Biol 2013; 992: 207–25. Balduini CL, Savoia A. Genetics of familial forms of thrombocytopenia. Hum Genet 2012; 131: 1821–32. Quiroga T, Goycoolea M, Munoz B et al. High prevalence of bleeders of unknown cause among patients with inherited mucocutaneous bleeding. A prospective study of 280 patients and 299 controls. Haematologica 2007; 92: 357–65. Watson SP, Lowe GC, Lordkipanidze M, Morgan NV. Genotyping and phenotyping of platelet function disorders. J Thromb Haemost 2013; 11(Suppl. 1): 351–63. Williams MS, Weiss EJ, Sabatine MS et al. Genetic regulation of platelet receptor expression and function: application in clinical practice and drug development. Arterioscler Thromb Vasc Biol 2010; 30: 2372–84. Kunicki TJ, Williams SA, Nugent DJ. Genetic variants that affect platelet function. Curr Opin Hematol 2012; 19: 371–9.
© 2014 John Wiley & Sons Ltd
DOI: 10.1111/hae.12414
REVIEW ARTICLE
von Willebrand disease and platelet disorders † S . J . I S R A E L S ‡ and S . A . B R O W N § ¶ E . J . F A V A L O R O , * I . B O D O, *Diagnostic Haemostasis, Haematology Department Institute of Clinical Pathology and Medical Research (ICPMR), Pathology West, Westmead Hospital, Westmead, NSW, Australia; †Department of Hematology and Stem Cell Transplantation, St. Istv an & St. L aszl o Hospital, Budapest, Hungary; ‡Department of Pediatrics and Child Health, University of Manitoba, Winnipeg, MB, Canada; §Department of Haematology & Haemophilia Centre, Royal Children’s Hospital; and ¶Department of Haematology, Pathology Queensland, Brisbane, Qld, Australia
Summary. The diagnosis and management of bleeding disorders is made difficult by the complexity and variety of disorders, clinical symptoms and bleeding type and severity. von Willebrand disease (VWD) and platelet disorders are disorders of primary haemostasis and together represent the most common inherited
bleeding disorders. In this article, we describe the diagnosis of VWD and platelet disorders and the treatment options for VWD.
Introduction
recognizes six subtypes of VWD [3]. Type 1 represents a partial quantitative deficiency of a functionally normal VWF protein. Type 3 VWD represents a severe (complete) deficiency of VWF. Type 2 VWD represents a group of qualitative VWF defects that comprise (i) type 2A VWD [loss of high molecular weight (HMW) VWF], type 2B VWD (enhanced functional binding of VWF to platelets that typically leads to loss of HMW VWF and mild thrombocytopenia), (iii) 2N VWD (loss of VWF-FVIII binding) and (iv) 2M VWF (VWF dysfunction not associated with loss of HMW VWF). The proper identification of VWD and its type is important as it has therapeutic implications [4]. In practice, VWD and its type can be determined by a process of laboratory testing that encompasses a comprehensive panel of different tests [1,5,6] (Table 1). The two main tests employed by virtually all laboratories are VWF antigen (VWF:Ag) and FVIII coagulant (FVIII:C); these, respectively, measure the level of VWF protein and FVIII activity. The most common VWF activity based test is the ristocetin cofactor (VWF:RCo) assay, which essentially measures VWF binding to the platelet VWF receptor GPIb. An additional test used by a proportion of laboratories is the collagen binding (VWF:CB) assay; collagen is a sub-endothelial matrix component which binds VWF in vivo. Additional ‘activity’ assays in general practice include those more recently released from commercial suppliers, such as the Werfen-IL and Siemens Innovance activity assays. VWF can also be assessed by other methods including multimer analysis to assess for loss of HMW VWF as well as structural abnormalities.
The diagnosis and management of von Willebrand disease (VWD) remains problematic for many laboratories and clinicians [1]. VWD arises from deficiency and/or defects of von Willebrand factor (VWF), a multimeric adhesive plasma protein essential for effective primary haemostasis. VWF is a multifunctional protein [2], which explains the heterogeneity in clinical symptoms and bleeding risk, as well as diagnostic challenges. Inherited platelet disorders include abnormalities of both number and function. Our understanding of specific rare platelet disorders has improved significantly in the last decade with the identification of specific disease-causing mutations. However, the investigation of individual patients with mild/moderate platelet disorders remains a challenge, as diagnostic tools available in most clinical laboratories often do not provide a definitive diagnosis. Improving our ability to define the abnormalities in common platelet disorders is our next challenge.
Challenges in the laboratory diagnosis of VWD The most recent classification scheme from the International Society on Thrombosis and Haemostasis Correspondence: Simon A. Brown, Department of Haematology, Pathology Queensland, Level 3 Block 7, Royal Brisbane & Women’s Hospitals, Herston, Brisbane, Qld 4029, Australia. Tel.: +617 3636 9846; fax: +617 3636 1552; e-mail: [email protected] Accepted 24 February 2014 © 2014 John Wiley & Sons Ltd
Keywords: desmopressin, platelet aggregation, platelet disorders, von Willebrand disease, von Willebrand factor
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Table 1. A practical guide to identification of von Willebrand disease and it’s type. VWD type
VWF:Ag
VWF:RCo
VWF:CB
FVIII
Multimers
1
↓ to ↓↓
↓ to ↓↓
↓ to ↓↓
N to ↓↓
2A
↓ to ↓↓
↓↓ to ↓↓↓
↓↓ to ↓↓↓
↓ to ↓↓
2B
N to ↓↓
↓ to ↓↓↓
↓ to ↓↓↓
N to ↓↓
2N
N to ↓↓
N to ↓↓
N to ↓↓
↓ to ↓↓↓
2M
↓ to ↓↓
↓ to ↓↓↓
↓ to ↓↓↓
↓ to ↓↓
No loss of HMW VWF; some multimer defects may however be observed
3
↓↓↓ (absent)
↓↓↓ (absent)
↓↓↓ (absent)
↓↓↓
No VWF present
Normal pattern but reduced intensity Loss of HMW VWF Loss of HMW VWF Normal pattern
RCo/Ag
CB/Ag
FVIII/Ag
>0.7
>0.7
>0.7
0.7
0.7
NA
Comments
2A and 2B VWD can only be distinguished by means of RIPA Similar to haemophilia A pattern; distinguish using VWF:FVIII binding assay or genetic analysis 2A and 2M VWD can only be distinguished by composite multipanel VWF testing Type 3 VWD can only be identified when VWF tests are sensitive to very low levels
Ag, antigen; CB, collagen binding; FVIII, factor VIIII; N, normal; NA, not applicable; RCo, ristocetin cofactor; RIPA, ristocetin induced platelet aggregation; VWF, von Willebrand factor.
In brief, type 1 VWD can be identified as a deficiency of VWF, with the level of deficiency correlating with the severity of the disorder. In these cases, low levels of VWF:Ag, VWF:RCo, VWF:CB and other activity assays (‘VWF:Act’) will be determined by laboratories testing patient plasma. However, as the VWF is functionally normal, similar (‘concordant’) levels of VWF will be identified using all VWF assays, and the ratio of any VWF assay to another will be close to one. In practice, a low level of VWF together with a ratio of VWF activity (VWF:RCo, VWF:CB or VWF:Act) to VWF:Ag above 0.7 is consistent with type 1 VWD (Table 1). In contrast, in type 2 VWD, VWF activity based assays will identify some VWF defect, with the defect identified helping to characterize the VWD type. Thus, loss of HMW VWF (present in 2A and 2B VWD) can be identified directly by multimer analysis or indirectly by a relatively larger reduction in VWF:RCo, VWF:CB and VWF:Act compared to VWF:Ag. This ‘VWF discordance’ can be expressed by a ratio of VWF activity to VWF:Ag below 0.5–0.7. Type 2M reflects a variety of functional defects, with most representing a platelet GP-Ib binding defect; hence VWF:RCo/VWF:Ag will usually be low, but VWF:CB/VWF:Ag may be normal. Type 2N VWD reflects a loss of VWF-FVIII binding; hence FVIII/VWF:Ag will be low, and phenotypically these patients resemble mild haemophilia A. The main problems relating to laboratory identification of VWD and its type are high inter-laboratory
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and inter-method assay variability, problems with lower limit of VWF detection, performance of insufficient test panels by laboratories to appropriately define all forms of VWD, and challenges in the interpretation of test findings. Thus, most laboratories struggle with differentiation between severe type 1 vs. 3 VWD, type 2M vs. 2A, 2M vs. 1, 2A vs. 2B, 2N vs. haemophilia A and type 3 VWD vs. haemophilia A. Severe type 1 and 3 VWD can only be distinguished if laboratories perform assays that are capable of detecting VWF levels down to 50 U dL 1 (>80–100 for critical bleeding or high-risk major surgery) for 7–14 days in case of major surgery and 1–5 days for minor procedures. A typical loading dose to achieve this is 50 VWF:RCo U kg 1 followed by 20–40 U kg 1 maintenance dose every 8–12 h for major surgery and 12–18 h for minor procedures. Daily VWF:RCo and FVIII determinations are mandatory to keep adequate levels of VWF and avoid ultra-high levels (i.e. >200 IU dL 1) of FVIII [9,20]. Standard thromboprophylaxis should be used in patients in whom VWF levels are normalized.
Management of VWD In general, three main modalities are available in our therapeutic armamentarium to stop bleeding in VWD patients: correction of the VWF plasma levels by (i) DDAVP or (ii) factor replacement, and (iii) decreasing the bleeding tendency by manipulating alternative routes with antifibrinolytic agents or oestrogen–progesterone drugs. These three approaches are not mutually exclusive.
Desmopressin (DDAVP) Desmopressin (1-deamino-8-d-arginine vasopressin) is a synthetic analogue of vasopressin [8,9]. DDAVP [Emosintâ (Kedrion, Pascoti Barga, Italia), Minirinâ (Ferring AB, Malmo, Sweden), Octostimâ (Ferring AB, Malmo, Sweden)] is inexpensive and devoid of any risk of transmitting blood-borne infections. It should be the preferred treatment modality, whenever possible. Usual administration is via the intravenous route, in a dosage of 0.3 lg kg 1, as an infusion over 20–30 min. A test infusion is recommended at the time of diagnosis to predict future response [10,11]. Desmopressin infusions are usually well tolerated, with tachycardia, headaches and flushing being the main side effects, usually ameliorated by slowing the infusion. Repeated DDAVP doses often become ineffective (a phenomenon called tachyphylaxis). This
Long-term secondary prophylaxis. Over the past decade, it has become clear that in severe forms of VWD, long-term prophylaxis is beneficial [21–23].
Adjunctive treatments by manipulating alternative routes of haemostasis Antifibrinolytic agents. As mucosal surfaces are rich in fibrinolytic activity [9], blocking fibrinolysis is a useful adjunctive measure to stop bleeding. Epsilon-aminocaproic acid (at a dose of 50–60 mg kg 1 every 4– 6 h) or tranexamic acid (at a dose of 10–15 mg kg 1
Table 2. Selected plasma-derived factor concentrates typically used for the treatment of VWD*. Product
Alphanate
Biostate
Fandhi
Manufacturer Purification
Grifols Heparin CTG
CSL Behring Precipitation + heparin CTG
Grifols Heparin CTG
Viral inactivation
S/D + dry heat (80°C, 72 h)
S/D + dry heat (80°C, 72 h)
FVIII IU mg 1 VWF:RCo/Ag VWF:RCo/FVIII
>100
50 0.94 1.21
0.8 2.0
Haemate P‡
Wilate
Wilfactin
Octapharma Ion ex. + size exclusion
LFB Ion ex. + affinity
S/D + dry heat (80°C, 72 h)
CSL Behring Multiple electrolyte precipitation Pasteurization (60°C, 10 h)
S/D + dry heat (100°C, 2 h)
>100
40
>80
S/D, 35 nm NF, dry heat (80°C, 72 h) >50† 0.95 >10
0.83 1.48
0.96 2.54
0.7 0.8–1.0
CTG, chromatography; Ion ex., ion exchange chromatography; S/D, solvent/detergent treatment; NF, nanofiltration. *Data from references no. 18 and 19. † Given as VWF:RCo/mg. ‡ Humate P in the US.
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every 8–12 h) may be administered orally, intravenously, or topically [9]. Oestrogen–progesteron preparations render the endometrium less susceptible to bleeding, and may be very useful in managing menorrhagia in VWD patients [8,9].
make it possible to use them as a screen prior to laboratory testing. However, existing tools have low specificity and will not provide a definitive diagnosis [27,28].
Investigation of platelet function abnormalities
Diagnostic approach to platelet disorders As there are no population-based data, the prevalence of inherited platelet disorders, which encompass both functional disorders and thrombocytopenia (Table 3), remains unknown. In studies of patients presenting with mucocutaneous bleeding, platelet abnormalities are at least as common as VWD. Severe disorders are often recognized in childhood, but mild disorders may go undiagnosed unless there is a family history that prompts testing, or until a haemostatic challenge results in significant bleeding. Algorithms have been developed to aid with the investigation of inherited platelet function disorders [24] (available at: www.ahcdc.ca/index.php/research/rare-inherited-bleeding-disorders) [25], and thrombocytopenias [26]. Validated bleeding assessment tools (BATs) are useful in standardizing information obtained from the patient history and accurately recording the severity and frequency of bleeding symptoms [27]. The high negative predictive value of some of these tools may
There is no ideal simple, inexpensive, sensitive screening test that reliably identifies patients requiring specialized testing of platelet function. Although both bleeding times and PFA-100/200â closure times have been used for this purpose, these tests are not adequately sensitive to rule out the need for further testing [29], and should be considered optional. A validated BAT may be more useful in assessing a patient’s bleeding propensity and determining whether further specialized laboratory investigations are warranted. The most widely used method for assessing platelet function is light transmission aggregometry (LTA), in which the change in optical density of a stirred sample of citrated platelet-rich plasma is measured by a photometer following the addition of agonists. Although many pre-analytical and analytical variables affect the results, and international surveys have shown that there is wide variation in methodology, LTA remains the gold standard platelet function test. Recommenda-
Table 3. Inherited platelet disorders. Inherited disorders of platelet function Abnormalities of receptors for adhesive proteins GPIb-IX-V complex (Bernard–Soulier syndrome*, platelet-type VWD*) GPIIb-IIIa (aIIbb3) (Glanzmann thrombasthenia) GPIa-IIa (a2b1) GPVI GPIV Abnormalities of receptors for soluble agonists Thromboxane A2 receptor P2Y12 receptor a2-adrenergic receptor Abnormalities of platelet granules d-granules (d-storage pool deficiency, Hermansky–Pudlak, Chediak–Higashi, TAR syndrome*) a-granules (Gray platelet syndrome*, ARC syndrome*, Quebec platelet disorder*, Paris–Trousseau–Jacobsen syndrome*) a- and d-granules (a,d-storage pool deficiency) Abnormalities of signal-transduction pathways Primary secretion defects Abnormalities of the arachidonic acid/thromboxane A2 pathway Gaq deficiency Partial selective PLC-b2 deficiency Defects in pleckstrin phosphorylation Defects in Ca2+ mobilization Abnormalities of cytoskeleton MYH9-related disorders (May–Hegglin anomaly, Sebastian syndrome, Fechtner syndrome, Epstein syndrome)* Wiskott–Aldrich syndrome* X-linked thrombocytopenia* Abnormalities of membrane phospholipids Scott syndrome*
Inherited thrombocytopenias Small platelets Wiskott-Aldrich syndrome† X-linked thrombocytopenia Normal-sized platelets Congenital amegakaryocytic thrombocytopenia Amegakaryocytic thrombocytopenia with radio-ulnar synostosis Thrombocytopenia with absent radii† Familial platelet disorder and predisposition to acute myeloid leukemia† Autosomal dominant thrombocytopenia Large platelets Bernard-Soulier syndrome† Velocardiofacial (22q deletion) syndrome Platelet-type von Willebrand disease† von Willebrand Disease Type 2B Gray platelet syndrome† ARC syndrome MYH9-related disorders† X-linked thrombocytopenia with thalassemia Paris–Trousseau–Jacobsen syndrome Dyserythropoietic anemia with thrombocytopenia
*Usually present with thrombocytopenia in addition to functional abnormalities. † May present with functional abnormalities in addition to thrombocytopenia.
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VWD AND PLATELET DISORDERS
tions for standardization have recently been published [30]. Whole blood aggregometry that measures aggregation as the change in electrical impedance between two electrodes, as platelets adhere and aggregate in response to agonists, has the advantages of using smaller blood volumes than LTA and requiring less manipulation of the sample. Lumi-aggregometry, in which the secretion of ATP from the dense granules is measured by the use of a luciferin/luciferase reagent, is useful in the identification of secretion defects. The specific cause of secretion defects remains unknown in most patients, but quantitation of platelet dense granules by whole mount electron microscopy will identify a subgroup with dense granule deficiency. Additional laboratory investigations including flow cytometry and mutation analysis can provide specific diagnoses in patients with defined abnormalities [29,31].
Investigation of thrombocytopenia Investigation of thrombocytopenia can be guided by platelet size (Table 3) [24,26,32]. Classification into small, normal-sized, or large platelets on the basis of mean platelet volume (MPV) should be confirmed by evaluation of the blood film. Automated cell counters underestimate platelet counts when platelet size is outside of the established reference range, and therefore should be combined with the evaluation of the peripheral blood film to provide additional information about platelet number, size, clumping and granularity and morphology of leucocytes and red cells [33]. Evaluation of the patient and family for evidence of clinical features in addition to the thrombocytopenia may identify a syndromic aetiology. Some inherited thrombocytopenias are also associated with platelet dysfunction (Table 3). Importantly, the genetic bases for more inherited thrombocytopenias have been identified in the last decade, allowing confirmation of the diagnosis in the patient and family members [32,34].
References 1 Favaloro EJ. Von Willebrand disease: local diagnosis and management of a globally distributed bleeding disorder. Semin Thromb Hemost 2011; 37: 440–55. 2 Yee A, Kretz CA. von Willebrand factor: form for Function. Semin Thromb Hemost 2014; 40: 17–27. 3 Sadler JE, Budde U, Eikenboom JCJ et al. 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. 4 Favaloro EJ, Franchini M, Lippi G. Biological therapies for von Willebrand disease. Expert Opin Biol Ther 2012; 12: 551–64.
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Phenotype/genotype correlations in mild platelet disorders Most individuals with platelet function disorders have abnormalities that are not clearly defined by standard clinical laboratory investigations. Some patients with mild/moderate bleeding and non-specific abnormalities of LTA have granule secretion defects [35], or subtle changes in receptor-mediated signal transduction. The investigation of these less well-defined abnormalities requires specialized testing, which may be beyond the capacity of most clinical laboratories. However, the definition of an individual family phenotype that directs sequencing of specific candidate genes has successfully identified previously unknown defects [36]. These are often heterozygous mutations that may represent only one of several abnormalities contributing to the bleeding phenotype; mild bleeding is likely to be a complex trait. The identification of subtle abnormalities as disease causing is complicated further by the fact that normal platelet reactivity is highly variable. Both hypo- and hyper-responsiveness to specific agonists have been shown to be associated with polymorphisms in genes encoding platelet adhesive protein receptors including integrins aIIbb3 and a2b1, and GPIb-IX-V [37]. More recently, genome-wide association studies have successfully identified proteins (some not previously suspected to be involved in platelet production or function) associated with variations in platelet count, MPV, and aggregation responses [38]. Study results vary depending on the phenotypic parameters chosen and the study population. However, these powerful tools are likely to change both our understanding of inherited platelet disorders and eventually how we investigate our patients with mucocutaneous bleeding.
Disclosures The authors stated that they had no interests which might be perceived as posing a conflict or bias.
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