A diagnostic approach to mild bleeding disorders

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1 Journal of Thrombosis and Haemostasis, 14: DOI: /jth REVIEW ARTICLE A diagnostic approach to mild bleeding disorders J. BOENDER, M. J. H. A. KRUIP and F. W. G. LEEBEEK Department of Hematology, Erasmus University Medical Center, Rotterdam, the Netherlands To cite this article: Boender J, Kruip MJHA, Leebeek FWG. A diagnostic approach to mild bleeding disorders. J Thromb Haemost 2016; 14: Summary. Mild inherited bleeding disorders are relatively common in the general population. Despite recent advances in diagnostic approaches, mild inherited bleeding disorders still pose a significant diagnostic challenge. Hemorrhagic diathesis can be caused by disorders in primary hemostasis (von Willebrand disease, inherited platelet function disorders), secondary hemostasis (hemophilia A and B, other (rare) coagulant factor deficiencies) and fibrinolysis, and in connective tissue or vascular formation. This review summarizes the currently available diagnostic methods for mild bleeding disorders and their pitfalls, from structured patient history to highly specialized laboratory diagnosis. A comprehensive framework for a diagnostic approach to mild inherited bleeding disorders is proposed. Keywords: bleeding disorder; bleeding score; blood coagulation disorders; diagnosis; DNA sequencing; hemostasis. Although patients with mild bleeding disorders may not often suffer from bleeding in daily life, problems may occur after a hemostatic challenge, including dental extractions or surgery. Menorrhagia is common in women with a bleeding disorder and a bleeding disorder is present in up to 17% of women with menorrhagia [4 6]. Diagnostic guidelines for mild bleeding disorders are still often based on expert opinion and experience, and can differ between countries. The difficulty of diagnosing mild bleeding disorders is exemplified by von Willebrand disease (VWD). The prevalence of VWD was reported to be 1.1 per persons in the 2013 annual report by the World Federation of Hemophilia (WFH), a number that is strikingly lower than the prevalence of 1% that has been reported in population studies [7,8]. In this review we discuss the currently available diagnostic techniques and propose a diagnostic approach to mild inherited bleeding disorders, as performed in our outpatient clinic. Patient and family history as a diagnostic tool Introduction Although the diagnosis of more severe bleeding disorders, such as moderate or severe hemophilia, is often well defined, the diagnosis of mild inherited bleeding disorders still poses a significant challenge to clinicians [1]. Bleeding symptoms also occur in otherwise healthy individuals, as over 20% of the general population report at least one bleeding symptom [2]. Therefore, both under- and overdiagnosis of mild bleeding disorders should be avoided. Patients with a bleeding disorder should have access to the proper medical treatment, but healthy individuals should not needlessly be exposed to potential complications of hemostatic therapy [3]. Correspondence: Frank W. G. Leebeek, Erasmus University Medical Center, PO Box 2040, 3000 CA Rotterdam, the Netherlands. Tel.: , fax: f.leebeek@erasmusmc.nl. Received 30 September 2015 Manuscript handled by: D. DiMichele Final decision: F. R. Rosendaal, 8 May 2016 When assessing a patient with a bleeding tendency it is crucial to obtain a detailed personal and family history, as is indicated in the algorithm in Fig. 1. Patients in whom bleeding has occurred since childhood and whose family members also suffer from bleeding are likely to have an inherited bleeding disorder. When abnormal bleeding occurs at a later age, or if there is a change in bleeding tendency, an acquired cause can often be identified. Many drugs have a deleterious effect on hemostasis, and detailed information on the use of medication should be obtained. The combined effects of drug use and a previously undetected inherited mild bleeding disorder may be the cause of abnormal bleeding. In autosomal dominant inherited disorders associated with bleeding, such as VWD or Ehlers Danlos syndrome (EDS), a positive family history of bleeding is often present. Incomplete penetrance can be seen in type 1 VWD, and a positive family history may even be absent in milder VWD cases [9]. Most autosomal recessive disorders have a negative family history, but bleeding may be reported in affected siblings. In families with hemophilia A or B, inheritance follows an X-linked pattern, with only male subjects being affected, although carriers may also have increased bleeding [10].

2 1508 J. Boender et al Patient with bleeding tendency Personal and family history ISTH-BAT Physical examination Abnormal PFA-100, VWF:Ag, VWF:RCo* or VWF:CB, or clinical suspicion of disorder in primary hemostasis Full blood count, liver and kidney function, PFA-100, VWF:Ag, VWF:RCo* and VWF:CB, APTT, PT, fibrinogen Abnormal APTT, PT, fibrinogen, or clinical suspicion of disorder in secondary hemostasis VWF assays >60 IU/dL: VWD unlikely - repeat testing VWF assays repeatedly >60 IU/dL: no VWD - test for IPFD - consider other disorder No abnormalities: Consider extended agonist panel, flow cytometry, electron microscopy Low platelet count: Thrombocytopenia - test for IPFD - consider 2B VWD, PT-VWD Blood smear, LTA + ATP release test/ lumiaggregometry If 1 abnormal aggregation: repeat test VWF levels IU/dL**: mild VWD/ low VWF - repeat testing If high BS: consider additional mild bleeding disorder If 2 abnormal aggregations, abnormal blood smear, impaired ATP release: IPFD VWF levels or activity <40 IU/dL**: VWD - repeat testing VWF activity/vwf:ag ratio >0.6: type 1 VWD VWF:Ag and activity < 5 IU/dL: type 3 VWD VWF activity/vwf:ag ratio <0.6: type 2 VWD: - multimer analysis - consider RIPA - consider mutation analysis Fibrinogen undetectable: afibrinogenemia Fibrinogen <1.0 g/l: hypofibrinogenemia -thrombin time and antigenic assay to test for hypodysfibrinogenemia - consider mutation analysis FVIII:C, FIX:C 40 U/dL and FXI:C 65% : - consider repeating FVIII:C APTT prolonged: - FVIII:C, FIX:C, FXI:C PT prolonged: - FVII:C FVIII:C < 40 U/dL: hemophilia A Or type 2N VWD FIX:C < 40 U/dL: hemophilia B - mutation analysis FXI:C < 65%: FXI deficiency PT and APTT prolonged: - FII:C, FV:C, FX:C If FV deficiency: - FVIII:C to exclude F5F8D No abnormalities: no IPFD - consider genetic testing - consider other disorder If abnormal: IPFD Consider extended agonist panel, flow cytometry, electron microscopy, mutation analysis for further classification No abnormalities in first analyses ISTH-BAT abnormal Children 3, men 4, women 6: Bleeding disorder more likely ISTH-BAT normal Children < 3, men < 4, women < 6: Low probability of bleeding disorder Consider disorder of vascular hemostasis Consider hyperfibrinolysis Consider HDCT: - refer to specialist - consider skin bleeding time for EDS - consider genetic testing Consider disorders of vascular formation: - refer to specialist Consider alpha 2 antiplasmin deficiency: - A2AP antigen and activity - consider genetic testing Consider PAI-1 deficiency: - PAI-1 antigen and activity - consider genetic testing No abnormalities detected Unclassified bleeding disorder? Consider repeating VWF:Ag and activity - FXIII:C Consider combined mild - consider repeating deficiency of FVIII:C, FIX:C coagulation factors, VWF and undetected mild IPFD Fig. 1. A diagnostic approach to mild inherited bleeding disorders. *There are more platelet-dependent VWF activity assays that are not necessarily inferior to the VWF:RCo assay, as is also stated by the SSC [38]; **Cut-off values differ between guidelines. ISTH-BAT, ISTH bleeding assessment tool; VWD, von Willebrand disease; IPFD, inherited platelet function disorder; PT-VWD, platelet-type von Willebrand disease; LTA, light-transmission aggregometry; BS, bleeding score; VWF:Ag, von Willebrand factor antigen; APTT, activated partial thromboplastin time; PT, prothrombin time; RIPA, ristocetin-induced platelet aggregation; F5F8D, combined FV and FVIII deficiency; A2AP, alpha 2 antiplasmin; PAI-1, plasminogen activator inhibitor-1; HDCT, hereditary disorders of connective tissue. Mucocutaneous bleeding is mainly characteristic of disorders of primary hemostasis. Late bleeding, that is, bleeding that occurs only several days after a challenge, occurs more often in disorders of fibrin formation or fibrinolysis. Paradoxically, some mild bleeding disorders are associated with thrombosis. If a patient with abnormal bleeding has had thrombosis or recurrent pregnancy loss, fibrinogen disorders and factor VII (FVII), FXI, FXIII, plasminogen activator inhibitor-1 (PAI-1) or alpha 2 antiplasmin (A2AP) deficiency may be the underlying cause [11,12]. Overall, bleeding symptoms are not specific for a certain underlying bleeding disorder and detailed laboratory testing is always needed for a definitive diagnosis. Bleeding scores as a diagnostic tool In the last decade there has been an increasing interest in using standardized diagnostic tools that systematically assess bleeding symptoms and aid in the diagnosis of bleeding disorders. Bleeding scores (BS) are questionnaires that systematically score each bleeding symptom according to the historically most severe bleeding episodes, using the sum of each individual symptom s score to aid in distinguishing between the absence and presence of a bleeding disorder. The first widely accepted BS was the Vicenza BS, developed by Rodeghiero et al. [2]. Many studies on bleeding scores in adults have used the MCMDM-1 VWD BS (also known as the Tosetto BS), which was derived from the Vicenza BS and was developed by the European Molecular and Clinical Markers for the Diagnosis and Management of type 1 von Willebrand disease (MCMDM-1 VWD) study [13]. This BS was condensed by Bowman et al. to decrease administration time [14]. The Pediatric Bleeding Questionnaire (PBQ), an adaptation of the MCMDM-1 VWD BS incorporating several pediatric-specific bleeding symptoms, has been developed for pediatric use [15]. The

3 Diagnosis of mild inherited bleeding disorders 1509 (condensed) MCMDM-1 VWD BS and PBQ have recently been reviewed [16]. These bleeding scores and their characteristics are shown in Table 1. In a recent prospective study of almost 800 VWD patients, a MCMDM-1 VWD BS >10 predicted future use of desmopressin or coagulant factor replacement therapy, regardless of VWD type or VWF levels [17]. This suggests that BS can also be used to predict future bleeding complications. The (condensed) MCMDM-1VWD scores have not only been used as a diagnostic tool for VWD, but also for a limited number of patients with other mild bleeding disorders, including coagulant factor deficiencies and hereditary hemorrhagic telangiectasia (HHT) [13,14,18,19]. In 2010, the ISTH bleeding assessment tool (ISTH-BAT) was developed by a joint working group of the Scientific and Standardization Committees (SSC) for VWF and perinatal/pediatric hemostasis of the ISTH, and is currently recommended in the diagnosis of bleeding disorders [20,21]. The ISTH-BAT, intended for use in both adults and children, scores 14 different bleeding symptoms from 0 to 4 (Table 1 and Table S1). The values for an abnormal ISTH-BAT score have been determined to be 3 in children, 4 for adult males and 6 for adult females [22]. Lowe et al. prospectively studied the ISTH-BAT in 79 patients suspected of having an inherited platelet function disorder (IPFD) and 21 healthy controls [23]. Although the BS was significantly higher in the patient group than in the control group (median 12 vs. 0, P < 0.05), it was not predictive for abnormalities on platelet function testing [23]. Bidlingmaier et al. prospectively studied the ISTH-BAT in 100 children with a suspected bleeding disorder, of whom 56 were eventually diagnosed with a bleeding disorder. The BS was higher in children with VWD and possible platelet function defects than in children who did not have a bleeding disorder identified. However, the BS was not higher in children with mild factor deficiencies (levels of FXIII, FVII, FX and FVIII ranging from 30% to 40%) compared with children with no identified bleeding disorder [24]. The currently used bleeding questionnaires are designed to be administered by a trained healthcare provider and can be time consuming. We have previously used a selfadministered MCMDM-1 VWD BS in a research setting, which gave similar results to the expert-administered version [25]. A self-administered version of the ISTH-BAT called the self-bat seems to be comparable to the expertadministered ISTH-BAT, particularly in women being assessed for von Willebrand disease, although relatively few patients were included in this study [26]. Physical examination Physical examination has limited use in the diagnosis of mild inherited bleeding disorders. Upon examination, Table 1 Vicenza-based bleeding scores and their characteristics Bleeding score Population Administered by Symptoms included Scores per symptom Normal range References Vicenza BS 2005 Adults Expert epi; cut; wound; oral; GI; muscle+joint; tooth; surg; mens; PPH MCMDM-1 VWD BS Or Tosetto BS 2006 Condensed MCMDM-1 VWD BS 2008 Pediatric Bleeding Questionnaire (PBQ) 2009 ISTH-BAT 2010 Children + adults 0 3 Men: < 3 Women: < 5 Adults Expert epi; cut; wound; oral; GI; tooth; surg; musc; joint; CNS; mens; PPH 1 to 4 All: < 4 [13] Adults Expert MCMDM-1 VWD BS 1 to 4 All: < 4 [14,18,19] Children Expert epi; cut; wound; oral; GI; tooth; surg; musc; joint; CNS; mens; PPH; other Expert epi; cut; wound; oral; GI; tooth; hem; surg; musc; joint; CNS; mens; PPH; other [2] 1 to 4 All: < 2 [15] 0 4 Children: < 2 Men: < 4 Women: < 6 Self-BAT 2015 Adults Patient Same as ISTH-BAT 0 4 Men: < 4 Women: < 6 [20,22 24] Category other includes umbilical stump bleeding, cephalohematoma, post-circumcision bleeding, post-venipuncture bleeding, macroscopic hematuria (PBQ) or umbilical stump bleeding, cephalohematoma, cheek hematoma caused by sucking during breast/bottle feeding, conjunctival hemorrhage or excessive bleeding following circumcision or venipuncture (ISTH-BAT and self-bat). epi, epistaxis; cut, cutaneous; wound, minor wounds; oral, oral cavity; GI, gastrointestinal tract; tooth, tooth extractions; surg, surgery; mens, menorrhagia; PPH, post-partum hemorrhage; musc, muscle hematoma; joint, hemarthrosis; CNS, central nervous system; hem, hematuria. [26]

4 1510 J. Boender et al bruises in different stages of resolution and petechiae can be seen. Sites where hematomas are frequent may show hyperpigmentation as a result of iron deposition. If bleeding is limited to one location, for example nose bleeds, a local cause of bleeding should be excluded. Several features may be identified in patients with connective tissue disorders. EDS patients often have skin hyperextensibility, widened atrophic scars on knees, shins and elbows, and joint hypermobility [27]. With increasing age, multiple telangiectasia can be identified in HHT patients, characteristically on or in the lips, oral cavity, fingers and nose [28]. Morphological abnormalities can be observed in patients with a bleeding tendency as part of a genetic syndrome, such as Noonan syndrome [21]. Laboratory approach to the diagnosis of mild bleeding disorders A full blood count should always be performed. Endocrine disorders, including thyroid disease and Cushing syndrome, and kidney or liver failure can cause an acquired bleeding disorder and may require laboratory investigation. The diagnostic approach to the patient suspected of having a bleeding disorder has been summarized in Fig. 1. First, global screening tests are performed, and based on these results more specific tests will be employed. Global diagnostic tests of hemostasis in patients with bleeding tendency In the first evaluation of a patient with a bleeding tendency, global tests of primary and secondary hemostasis are performed. The global coagulation assays that are used most often are the prothrombin time (PT) and activated partial thromboplastin time (APTT), which give information on most coagulation factors (Fig. 1). The PT is prolonged in FVII deficiency, the APTT in FVIII, FIX and FXI deficiency and both PT and APTT may be prolonged in the case of deficiency of FII, FV and FX, and very low fibrinogen levels. The APTT may also be prolonged if there is FXII deficiency or lupus anticoagulant (both not associated with bleeding) [29]. Both the PT and APTT can be prolonged in liver failure, vitamin-k deficiency or use of vitamin K antagonists [30]. However, a normal PT and/ or APTT does not exclude mild coagulant factor deficiency [31]. Deficiency of FXIII is not detected by PT or APTT. Other global tests such as thromboelastography and thrombin generation have been in use for many years and have recently gained increasing interest [32,33]. However, their usefulness as a diagnostic tool for mild bleeding disorders is not well known yet. Skin bleeding time according to Ivy is considered obsolete as a test of primary hemostasis and has been replaced by automated platelet function tests, including the platelet function analyzer (PFA-100 Ò ), in many laboratories. The PFA-100 Ò measures the ability of platelets to adhere and aggregate under high shear stress to a membrane covered with collagen and epinephrine or collagen and ADP [34]. PFA-100 Ò is influenced by hematocrit, VWF activity, low platelet count and platelet dysfunction. An abnormal PFA-100 Ò should prompt further investigation of VWD and inherited platelet function disorders (IPFDs) [35]. Although PFA-100 Ò can be a useful screening tool for some IPFDs and more severe VWD, its sensitivity is only 61 71% for VWD and 24 58% for IPFDs [36,37]. A normal PFA-100 Ò does not exclude mild VWD or IPFD, and is therefore not recommended as a screening test for IPFDs by the ISTH SSC on platelet physiology [21]. Global fibrinolysis assays are mainly used in research and are not readily available in clinical practice. Specific diagnostic laboratory testing in the event of suspicion of disorders of primary hemostasis Von Willebrand disease The laboratory diagnosis of VWD is based on VWF antigen concentration (VWF:Ag) and VWF function tests, combined with FVIII coagulant activity (FVIII:C) [9]. The mainstay for measuring VWF function is still the VWF ristocetin cofactor assay (VWF: RCo), which measures platelet agglutination in the presence of VWF induced by ristocetin. However, this test has a high coefficient of variation, which can lead to both false and missed diagnosis of VWD when an individual has borderline VWF levels [38]. In addition, several VWF gene polymorphisms cause a falsely reduced VWF:RCo and may therefore lead to misclassification of type 1 VWD as type 2 [39]. In the near future, other assays will probably replace the VWF:RCo as the reference standard for measuring VWF platelet-dependent activity [38,40]. In our laboratory we use the VWF:GPIbM assay, which uses a recombinant mutant gain-of-function GPIb fragment that spontaneously binds VWF. The VWF:GPIbM assay is easy to use and provides swift, precise and sensitive results [38,40]. Because the VWF:GPIbM assay does not require ristocetin, it is not subjective to the VWF polymorphisms that affect the VWF:RCo assay [38]. Adding collagen binding assays (VWF:CB) to the diagnostic workup has been shown to increase the diagnostic rate of VWD by as much as 30% [41]. VWF:CB is also able to detect subtypes with defective collagen binding and is often used to aid in the classification of VWD [42]. For more details on the diagnostic approach to, and the classification of, VWD, we refer to a recent review by de Jong and Eikenboom [40]. Although the diagnostic criteria and clinical relevance of moderate and severe VWD are well established, there is still debate about the clinical relevance of mildly decreased VWF levels. The current ISTH guidelines for the diagnosis of VWD do not clearly mention a cut-off value for VWF: Ag or VWF:RCo or another VWF activity test for the diagnosis of VWD, and these values vary between

5 Diagnosis of mild inherited bleeding disorders 1511 countries and research groups [9,43 46]. VWF levels are usually considered to be abnormal when they are below the 2.5th percentile of the normal population, usually IU dl 1 for both VWF:Ag and VWF:RCo, depending on ABO blood group distribution [41,47]. VWF levels below IU dl 1 are typically associated with hemorrhagic diathesis and often co-segregate in affected families, but this is much less clear for marginally low (40 60 IU dl 1 ) VWF levels [13,48]. This group is often seen more as having a risk factor for bleeding, referred to as low VWF, and not as having VWD [49]. Nevertheless, studies have demonstrated increased bleeding in people with marginally low VWF levels and low VWF may be considered a mild bleeding disorder [50 52]. VWF levels are influenced by a number of endogenous and exogenous factors and display a large intrapersonal variability [53]. Repeated measurement of VWF should be performed to minimize the influence of environmental factors such as stress, physical exercise and inflammatory disease, the lowest levels measured probably best reflecting the resting situation in the patient. VWF levels are 25% lower in blood group O than non-o, and because VWF levels increase with age in both VWD patients and the general population, aging patients may no longer meet the diagnostic criteria for VWD [54]. Although mutation analysis of the VWF gene can be valuable for the classification of more severe subtypes (especially types 2B and 3 VWD), mutation analysis currently has no place in the diagnosis of mild VWD [55]. Inherited platelet function disorders The diagnosis of IPFDs is complex and a wide range of laboratory approaches for investigating IPFDs are used. The ISTH SSC on platelet physiology recommends performing a blood smear, light transmission aggregometry (LTA) with a limited number of agonists, assessment of platelet granule release and the analysis of major surface glycoproteins by flow cytometry (see Fig. 1) [21]. Light transmission aggregometry allows assessment of various platelet functions and is the preferred laboratory test in the diagnosis of IPFDs [56,57]. In a prospective study of 229 patients analyzed for the presence of a bleeding disorder and 105 healthy controls, abnormal LTA was more likely in patients with IPFDs than healthy controls. However, LTA had only moderate sensitivity, because 47% of IPFD patients had normal LTA results [58]. Despite being highly reproducible, repeated LTA testing is recommended to exclude falsely abnormal platelet aggregation as a result of pre-analytical and analytical variables, especially if only one agonist reveals abnormalities [59,60]. Assays that measure ATP or serotonin release from platelet granules may detect IPFDs not detected by LTA [60]. Lumiaggregometry seems to be highly specific, although less sensitive for IPFDs even when patients have normal LTA [61]. The limitations of the assay include a lack of specificity for different concentrations of ADP and of sensitivity according to recent publications [61,62]. LTA and lumiaggregometry have been more extensively reviewed by Cattaneo [63]. Abnormal aggregation in LTA or ATP release to a specific agonist correspond to different categories of IPFD, but are beyond the scope of this review. Platelet whole mount electron microscopy evaluates the number of dense granules and can be used to exclude dense granule deficiency, but has only limited availability. Flow cytometry allows assessment of many platelet characteristics. Although it can provide a swift diagnosis of IPFDs and also has applications outside diagnosing IPFDs, flow cytometry is expensive and still lacks international standardization [64]. The genetic background of IPFDs is diverse, and many genes affecting platelet function have been identified in recent years [65]. Although large gene panels are able to detect genetic defects in IPFD patients, mutation analysis is currently only of limited use in the diagnosis of IPFDs [66]. A more detailed review including recommendations for the diagnostic approach to IPFDs by the ISTH SSC on platelet physiology has recently been published by Gresele et al. [21]. Specific diagnostic laboratory testing in the event of suspicion of disorders of secondary hemostasis A mild to moderate deficiency of any of the coagulation factors may result in a mild bleeding phenotype. The diagnostic approach in the event of abnormal APTT and/ or PT is given in Fig. 1. The most frequently occurring deficiencies are of FVIII or FIX, as observed in hemophilia A or B. These levels are measured by specific factor tests, including one-stage or chromogenic assays. Importantly, recent studies have reported a considerable discrepancy in measured FVIII:C between these assays in up to 40% of mild hemophilia A patients, caused by the different properties of these patients mutant FVIII protein [31,67,68]. This can lead to a missed diagnosis of mild hemophilia A, which might be avoided by using multiple assays in the diagnostic process of HA. FVIII is bound to VWF in the circulation and is influenced by the same genetic and environmental factors as VWF. Low FVIII:C may also be caused by low VWF levels, as found in type 1 or 3 VWD, or in type 2N VWD, which is characterized by impaired VWF to FVIII binding. As in mild VWD, repeated FVIII:C measurements should be performed when mild HA or a carrier of hemophilia is suspected. Both mild HA and mild HB are defined as FVIII:C or FIX:C > 5 40% of normal (see Table 2) [69]. Determination of FVIII:C or FIX:C should be performed in hemophilia carriers, but carriers may have an increased bleeding tendency even when factor levels are normal [10]. Mutation analysis of the F8 or F9 genes is widely performed for genetic counselling and can confirm carriership.

6 1512 J. Boender et al Determination of FII:C, FV:C, FVII:C, FX:C, FXI:C and FXIII:C can be performed using a one-stage or chromogenic assay, but for FII deficiency, an antigenic assay should also be performed to exclude dysprothrombinemia [11, 70 76]. In FV-deficient patients, FVIII:C should also be measured to exclude combined FV and FVIII deficiency (F5F8D) [77]. Disorders of fibrinogen are classified in afibrinogenemia, hypofibrinogenemia and dysfibrinogenemia [11,78]. Fibrinogen is usually measured by the Clauss assay, which measures the level of functional fibrinogen. Fibrinogen levels are lower than 1.0 g L 1 in hypofibrinogenemia or hypodysfibrinogenemia and undetectable in afibrinogenemia. Thrombin time and antigen assay of fibrinogen should also be performed to assess the likelihood of dysfibrinogenemia [11,79]. An accurate diagnosis of dysfibrinogenemia can only be made by identifying the causative genetic defect. The bleeding phenotype is strongly associated with coagulant activity levels of fibrinogen, FX and FXIII, but this association is only poor for FV and FVII deficiencies, whereas in FXI deficiency, there is hardly an association between factor level and bleeding severity [80]. In 2012, the Rare Bleeding Disorders Working Group of the ISTH SSC for FVIII and FIX proposed a set of laboratory criteria for the diagnosis and classification of inherited deficiency of fibrinogen, FII, FV, FVII, FX, FXIII and combined FV and FVIII deficiency, which are summarized in Table 2 [77]. Specific diagnostic laboratory testing in the event of suspicion of other bleeding disorders Hyperfibrinolysis Little information is available on the diagnosis of the rare fibrinolysis disorders. Global clot lysis assays have been developed, but have so far only Table 2 Classification of disorders of secondary hemostasis Disorder Disease severity Severe Moderate Mild Coagulant factor activity Hemophilia < 1% 1 5% > 5 < 40% A&B Fibrinogen Undetectable gl 1 >1gL 1 Factor (F) II Undetectable 10% > 10% FV Undetectable < 10% 10% FV + FVIII < 20% 20 40% > 40% FVII < 10% 10 20% > 20% FX < 10% 10 40% > 40% FXIII Undetectable < 30% 30% FXI Mild moderate < 20% Usually asymptomatic 20 65% References: hemophilia A and B [69], other disorders [77,80]. been used in a research setting and are not yet used in diagnostic algorithms. The diagnosis of PAI-1 deficiency is challenging. Tests that measure PAI-1 antigen do not detect qualitative defects. Activity tests are not sensitive when PAI-1 levels are low, and have been reported to fail to detect PAI-1 activity in the normal range [81]. Both A2AP antigen and function should be measured for the diagnosis of A2AP deficiency, which in heterozygotes may result in a mild bleeding phenotype [82,83]. Disorders of vascular hemostasis Assays that measure primary and secondary hemostasis are usually normal in hereditary disorders of connective tissue (HDCT), although the bleeding time may be prolonged in EDS, with abnormal bleeding times being reported in % of HDCT patients [27,84,85]. The diagnosis of EDS can be confirmed by biochemical analysis of collagen types I, III and V. The diagnosis of EDS and the other HDCT can also be confirmed by identifying the genetic defect [27]. The most prevalent disorder of vascular formation is hereditary hemorrhagic telangiectasia (HHT, also known as Osler Weber Rendu syndrome), affecting 1 in individuals. HHT does not lead to abnormal test results of primary or secondary hemostasis [28]. The diagnosis is based on clinical findings, and is considered definite when at least three of the following criteria are present: spontaneous and recurrent epistaxis, multiple cutaneous telangiectasia, visceral involvement and a first-degree relative with HHT [28]. Genetic testing can confirm the diagnosis but is complex because multiple genes are implicated in HHT and a mutation cannot always be identified [28]. Our diagnostic approach to mild bleeding disorders Based on the above-mentioned considerations and recommendations we use the following diagnostic approach in patients referred to our department, as outlined in Fig. 1. Our first step is to obtain both the family and a detailed personal (bleeding) history, followed by a physical examination. We have integrated the ISTH-BAT score in our electronic patient file and every doctor calculates this when a patient suspected of a (mild) bleeding disorder is referred. Although the use of this BS for diagnosing or excluding a mild bleeding disorder has not yet been validated, it allows for a structured and standardized approach to bleeding symptoms and normal values have been established (Table 1) [22]. It is still not known whether the BS can be used to discriminate between the absence and presence of mild bleeding disorders [19,24]. Therefore, irrespective of the ISTH-BAT score, we perform an initial limited laboratory evaluation, including a full blood count, PFA-100 Ò, VWF:Ag, VWF:GPIbM, VWF:CB, APTT, PT and fibrinogen. We perform additional assays based on both the bleeding history and the outcome of the screening assays. Despite its known low sensitivity and specificity, we still

7 Diagnosis of mild inherited bleeding disorders 1513 use the PFA-100 Ò as an indicator of the likelihood of IPFDs. In the event of an abnormal PFA-100 Ò result and a normal platelet count and VWF levels, we always perform LTA. Because the PFA-100 Ò is known for its low sensitivity for mild IPFD, a normal result by no means excludes IPFDs. In the event of a normal PFA-100 Ò result we still perform LTA if no other bleeding disorder is diagnosed in a subset of referred patients, for instance in patients with high BS. In the case of an abnormal secondary hemostasis test (fibrinogen, APTT or PT), we will perform specific factor tests, as specified in Fig. 1. In the case of normal screening test results and a normal BS, in most patients no further tests are performed. However, if an individual is referred because of a familial bleeding disorder (e.g. VWD), who has had no hemostatic challenges, we repeat the measurements. In the case of normal laboratory findings, but with a high BS, additional tests will be performed, including repeated VWF measurements and FXIII and A2AP activity (Fig. 1). An isolated prolonged bleeding time may help diagnose a disorder of vascular hemostasis and is performed in patients with symptoms of hypermobility or skin abnormalities. Because mildly reduced factors VIII and IX may be seen in individuals with normal APTT, we also measure these factors in individuals with a marked bleeding tendency. A combination of mild deficiencies or abnormalities should also be considered. Despite our efforts, also in our department we are not able to make a diagnosis of a bleeding disorder in around half of the patients. The question remains whether these patients should be treated before surgical or dental interventions. Especially in patients with a high BS and in those with previous bleeding after surgical interventions, we recommend using tranexamic acid and/or desmopressin peri-operatively. This has recently been studied by Obaji et al., who applied hemostatic therapy consisting of desmopressin and/or tranexamic acid to a large group of patients with bleeding diathesis without any laboratory diagnosis and found no bleeding in 90% of patients undergoing an intervention [86]. Another problem arises in patients who have been diagnosed with a mild bleeding disorder (e.g. VWD type 1) in the past, who have normal results on repeated testing [87]. It is well known that VWF levels increase with aging, which may lead to normalization of VWF levels [54]. It was recently shown that these historical type 1 VWD patients do not have a different bleeding score to VWD patients with low VWF levels, and we currently still treat these patients preoperatively [54,87]. Future directions The diagnosis of mild bleeding disorders is based on clinical and laboratory testing, but laboratory results are not able to reliably predict bleeding. Excluding a disorder can be especially challenging when a patient has borderline test results or when the medical history is highly suggestive of a bleeding disorder and repeated laboratory testing does not show any abnormalities. Genetic testing is becoming increasingly available and affordable with the introduction of next-generation sequencing. However, the clinical relevance of many genetic variants is not well known, and can be subject to differences between ethnicities. For example, VWF mutations in VWD patients of European ancestry are commonly found in healthy people of African ancestry [88]. Often no mutation is identified in patients with an inherited bleeding disorder. More studies on the genetic background of (mild) inherited bleeding disorders are ongoing and have already identified a number of genetic defects [89]. Conclusion The diagnosis of mild inherited bleeding disorders requires a detailed patient and family history, with the newly developed bleeding scores as a valuable diagnostic tool, followed by specific laboratory testing. Despite these improvements, mild bleeding disorders still pose a significant diagnostic challenge. This may be improved by testing at the molecular level, but studies linking bleeding phenotype with genetic variants are needed. Addendum J. Boender and F. W. G. Leebeek wrote the manuscript. M. J. H. A. Kruip wrote and critically revised the manuscript. Disclosure of Conflict of Interests M. J. H. A. Kruip reports grants from Ferring, Pfizer and ZonMW (Dutch Organisation for Health Research and Development), outside the submitted work. F. W. G. Leebeek reports grants from CSL Behring, Baxter and the Dutch Hemophilia Foundation (Stichting Hemophilia), and is a consultant for UniQure, for which fees go to the ErasmusMC, outside the submitted work. Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1 ISTH-BAT scoring key. References 1 Pereira J, Quiroga T, Mezzano D. Laboratory assessment of familial, nonthrombocytopenic mucocutaneous bleeding: a definitive diagnosis is often not possible. Semin Thromb Hemost 2008; 34: Rodeghiero F, Castaman G, Tosetto A, Batlle J, Baudo F, Cappelletti A, Casana P, De Bosch N, Eikenboom JC, Federici AB,

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