Laboratory Diagnostics of Inherited Platelet Disorders

Home , Gray platelet syndrome, TAR syndrome

DOI 10.1515/cclm-2014-0131 Clin Chem Lab Med 2014; 52(8): 1091–1106

Review

Cecilia Carubbia, Elena Massellia, Antonio Nouvenne, Domenico Russo, Daniela Galli, Prisco Mirandola, Giuliana Gobbi* and Marco Vitale* Laboratory diagnostics of inherited platelet disorders

Abstract: Inherited platelet disorders (IPDs) are the gen- Introduction eral and common denomination of a broad number of different rare and congenital pathologies affecting plate- Inherited platelet disorders (IPDs) are rare, congenital dis- lets. Even if these disorders are characterized by widely eases of platelet number and/or function characterized by a heterogeneous clinical presentations, all of them are com- widely heterogeneous clinical presentation and a challeng- monly present as defects in hemostasis. Platelet num- ing diagnostic work-up. They usually present as primary ber and/or function are affected by a wide spectrum of hemostatic defects that may range from a prolonged bleed- severity. IPDs might be associated with defects in bone ing after trauma or surgery in adulthood to life-threatening, marrow megakaryocytopoiesis and, rarely, with somatic spontaneous hemorrhages (gastrointestinal, genitourinary defects. Although in the last few years new insights in the or even intracranial) occurring early in life [1, 2]. genetic bases and pathophysiology of IPDs have greatly Defects in platelet function are frequently accom- improved our knowledge of these disorders, much effort panied by abnormalities in bone marrow megakaryocy- still needs to be made in the field of laboratory diagnosis. topoiesis, resulting in reduced platelet counts and altered This review discusses the laboratory approach for the dif- platelet morphology. ferential diagnosis of the most common IPDs, suggesting a In some cases, platelet disorders are associated with common multistep flowchart model which starts from the somatic defects, such as reduced or delayed pigmentation simpler test (platelet count) ending with the more selec- Hermansky-Pudlak syndrome (HPS), hearing loss, renal tive and sophisticated analyses. impairment and/or cataracts (MYH9-related diseases), eczema and susceptibility to infections Wiskott-Aldrich Keywords: bleeding; laboratory tests; platelets; rare syndrome (WAS), limb abnormalities (thrombocytopenias diseases. associated with skeletal defects). While the process of IPDs characterization significantly aCecilia Carubbi and Elena Masselli contributed equally to this work. contributed to our understanding of the genetic bases of *Corresponding authors: Giuliana Gobbi, PhD, Department of megakaryocytopoiesis and platelet function, unfortunately Biomedical, Biotechnological and Translational Sciences (S.Bi. little progress has been made in the field of laboratory diag- Bi.T.), University of Parma, Ospedale Maggiore, Via Gramsci, 14, nostics of these diseases. In fact, diagnosis of the majority 43100 Parma, Italy, Phone: +39 0521 033142, Fax: +39 0521 033033, E-mail: [email protected]; Marco Vitale, MD, Department of IPDs still primarily relies on personal and family history, of Biomedical, Biotechnological and Translational Sciences (S.Bi. co-morbidities (i.e., skeletal defects), peripheral blood Bi.T.), University of Parma, Ospedale Maggiore, Via Gramsci, 14, smear analysis and genetic testing, when available [3, 4]. 43100 Parma, Italy, Phone: +39 0521 033032, This review will discuss the diagnostic algorithm – Fax: +39 0521 033033, E-mail: [email protected] focusing on laboratory tests – for the most common IPDs; Cecilia Carubbi, Elena Masselli, Daniela Galli and Prisco Mirandola: diseases are presented on the basis of their involvement of Department of Biomedical, Biotechnological and Translational Sciences (S.Bi.Bi.T.), Anatomy and Histology Unit, University of platelet count, function, or both. Parma, Ospedale Maggiore, Parma, Italy Antonio Nouvenne: Department of Clinical and Experimental Medicine, University of Parma, Ospedale Maggiore, Parma, Italy Disorders of platelet number Domenico Russo: Unit of Blood Diseases and Cell Therapies, University of Brescia, AO Spedali Civili Brescia, Brescia, Italy This group encompasses MYH9-related disorders, congenital amegakaryocytic thrombocytopenia (CAMT), 1092 Carubbi et al.: Inherited platelet disorders diagnosis thrombocytopenias associated with skeletal defects, Platelet count is usually below 50 × 109/L and their size is X-linked thrombocytopenia (XLT) with dyserythropoiesis, normal [10]. familial platelet disorders with predisposition to acute Bone marrow aspirate reveals marked megakaryo- leukemia (FDP/AML), benign mediterranean macrothrom- cytic hypoplasia that may progress to bone marrow bocyopenia (BBM), familial thrombocytopenia 2 (THC2). failure. Mutations of the thrombopoietin (TPO) recep- Genetic analysis is the sole specific laboratory test for tor (c-mpl) underlie this disease and can be detected by these IPDs and is currently clinically available for CAMT, genetic testing [11]. thrombocytopenia with absent radii (TAR), thrombocytopenia with radio-ulnar synostosis (ATRUS), XLT with dyserythropoiesis and FDP/AML [5] (Table 1). Thrombocytopenias associated with skeletal Beyond genetic testing, diagnosis is essentially based defects on family history (usually relevant in autosomal dominant disorders such as MYH9-related disorders, ATRUS, This group of IPDs encompasses three different thrombo- Di George/Velocardiofacial syndrome (VCFS), thrombocytopenias associated with peculiar skeletal defects: TAR, cythopenia with predisposition to leukemia, BBM, THC ATRUS and Di George/VCFS. 2) and presence of patognomonic-associated defects TAR is an autosomal recessive disease that manifests affecting other organs (see Figures 1 and 2). During the early in life with a bleeding diathesis that tends to amelio- laboratory diagnostic work-up, a key role is detained by rate with time due to a progressive increase of the platelet peripheral blood smear analysis for platelet morphology count. Diagnosis is straightforward given the presence (i.e., presence of macrothrombocytes – as in the case of of the typical bilateral radial anomalies that affect TAR MYH9-related disorders and BMM – or detection of spin- patients. Bone marrow megakaryocytes are reduced, but dle-shaped Döhle leukocyte inclusions, as in the case of platelet size and function are normal. Defects in the TPO/ MHY9-related diseases) [6]. c-mpl signaling may be found with appropriate genetic tests [12]. ATRUS is an autosomal dominant amegakaryocitic MYH9-related disorders thrombocytopenia associated with aplastic anemia, radio-ulnar synostosis, clinodactyly, syndactyly, hip- This term indicates a group of autosomal dominant dysplasia and sensorineural hearing loss. Like TAR, thrombocytopenias (May-Hegglin anomaly, Sebastian also ATRUS presents with severe thrombocytopenia Syndrome, Fechtner Syndrome and Epstein syndrome) at birth that may improve with time [13]. Detection of caused by mutations within the MYH9 gene, encoding for mutations of HOXA11, a member of the homeobox genes the non-muscle myosin IIA heavy chain which is part of encoding for proteins that detain a central role in skel- the contractile cytoskeleton of megakaryocytes, platelets etal development and hematopoiesis, allows final diag- and neutrophils [7, 8]. nosis [14]. As a result, bone marrow megakaryocitopoiesis is Di George/VCFS is an autosomal dominant macro- ineffective and platelets display abnormally larger dimen- thrombocytopenia typified by cardiac abnormalities, cleft sions (macrothrombocytes). Platelet aggregation tests palate, thymic aplasia, mental retardation, disimmunity. are normal. Thrombocytopenia ranges from 20 × 109/L to The gene involved, usually with a hemizygous loss of 130 × 109/L, but bleeding phenotype is usually mild. Diag- allele, is GP1BB, located at 22q11, one of the three genes nosis is suggested by peculiar co-morbidities, such as sen- defective in Bernard-Soulier syndrome (BSS). Thus, clini- sorineural hearing loss, cataracts, glomerulonephritis, cal manifestations are usually mild and may overlap with but definitive identification of this IPD requires the dem- heterozygous BSS. Again, clinical diagnosis is suggested onstration of a causative mutation within MHY9 [9]. by the relevant co-morbidities and requires confirmation by genetic testing [15].

Congenital amegakaryocytic thrombocytopenia (CAMT) X-linked thrombocytopenia with dyserythropoiesis CAMT is an autosomal recessive disorder characterized by symptomatic thrombocytopenia manifesting early This X-linked platelet disorder is due to mutations of the in life, occasionally with a severe bleeding phenotype. GATA1 gene, encoding for the transcription factor GATA1, Carubbi et al.: Inherited platelet disorders diagnosis 1093

Table 1 Classification of inherited platelet disorders and laboratory tests currently available.

Gene (location) Inheritance Genetic testing Lab test(s) clinically available

MYH9-related disorders MYH9 (22q12-13) AD Yes PLT count Peripheral blood smear Platelet aggregometry Congenital amegakaryocytic c-MPL (1p34) AR Yes PLT count thrombocytopenia Peripheral blood smear Bone marrow aspirate Thrombocytopenias associated Y19 (1q21.1), HOXA11 (7p15-p14.2), AD/AR Yes PLT count with skeletal defects GPIBB (22q11) (Bone marrow aspirate) X-linked thrombocytopenia with GATA1 (Xp11.23) X-linked Yes PLT count dyserythropoiesis Peripheral blood smear Bone marrow aspirate Familial platelet disorders with RUNX1 (21q22) AD Yes PLT count predisposition to leukemia Peripheral blood smear Platelet aggregometry Benign mediterranean GPIBA AD No PLT count macrothrombocyopenia Peripheral blood smear Familial thrombocytopenia 2 ANKRD26, MASTL, ACBD5 AD No PLT count Bone marrow aspirate Bernard-Soulier syndrome GPIBA (17p13) AR Yes PLT count GPIBB (22q11) Bleeding time Closure time Peripheral blood smear Platelet aggregometry Flow cytometry Glanzmann Thromboasthenia ITGA2B, ITGB3 (17q21-23) AR No PLT count Bleeding time Closure time Peripheral blood smear Platelet aggregometry Flow cytometry Platelet-type von Willebrand GPIBA (17p13) AD Yes PLT count disease Bleeding time Closure time Peripheral blood smear Platelet aggregometry Protein electrophoresis Wiskott-Aldrich syndrome WAS X-linked Yes PLT count Bleeding time Closure time Peripheral blood smear Storage pool diseases LYST (1q42.1-42.2), HPS1, AP3B1, AD/AR No PLT count HPS3, HPS4, HPS5, HPS6, DTNBP1, Bleeding time HPS8, FLI1 (11q23) Closure time Peripheral blood smear Platelet aggregometry Flow cytometry Electron microscopy Scott syndrome ABCA1 AR No PLT count Bleeding time Closure time 1094 Carubbi et al.: Inherited platelet disorders diagnosis

(Table 1 Continued)

Gene (location) Inheritance Genetic testing Lab test(s) clinically available

Peripheral blood smear Platelet aggregometry Flow cytometry

Agonist PLT receptor defects TBXA2R, P2Y12 AD/AR No PLT count Bleeding time Closure time Peripheral blood smear Platelet aggregometry

whose role in both eryhtroid and megakaryocytic differ- asymptomatic. Peripheral blood smear reveals large size entiation of hematopoietic progenitors is well established. platelets with a conserved functionality in in vitro studies. As a result, patients present with marked thrombocy- Bone marrow megakaryocytes are normal. No genetic test topenia sustained by a defective megakaryocitopoiesis. is currently available [20, 21]. Platelets are large with a decreased granular content. Dyserythropoiesis usually accompanies megakaryocytic defects and may account for transfusion dependence. Due Familial thrombocytopenia 2 (THC2) to the complexity and heterogeneity of the disease, diagnosis may be challenging and requires demonstration of THC2 patients present with a mild to moderate thrombo- GATA1 mutation(s) [16]. cytopenia that becomes usually symptomatic only during hemostatic stress (trauma, major surgery). Peripheral blood smear is not helpful since platelet size is normal Familial platelet disorders with predisposi- although bone marrow megakaryocytes show abnormal tion to acute leukemia (FDP/AML) morphology, typified by a smaller size and hypolobated nuclei [22]. Several gene mutations have been described This is a rare, autosomal dominant condition sustained and can be screened to rule out other normothrombocytic by mutations in the RUNX1 gene, encoding for a key tran- thrombocytopenias [23–25] (see Table 1). scription factor in hematopoiesis. Haploinsufficiency of RUNX1 accounts for inadequate transcription of genes involved in megakaryocytic differentiation [17]. Although platelet count is only moderately reduced Disorders of platelet function (and (80–150 × 109/L), patients may experience severe bleeding number) diathesis due to aspirin-like platelet dysfunction [18, 19]. Consequently, laboratory diagnosis, beyond genetic Among inherited platelet disorders, platelet function dis- testing, requires platelet aggregometry. orders are characterized by normal or reduced platelet Involvement of RUNX1 in the disease pathogenesis number (reference range 140–400 × 109/L) associated with may explain the tendency to develop hematologic malig- impaired physiological mechanisms of activation and nancies, such as myelodisplastic syndrome and acute aggregation. Main causes are represented by: dysfunc- myeloid leukemia. tions of surface platelet receptors, deficiency of platelet granule content and abnormalities of degranulation. Diagnosis of platelet function disorders is initially Benign mediterranean macrothrombocyto- based on traditional laboratory tests including bleeding penia (BMM) time, platelet aggregometry and platelet function analyz- ers (PFA) (see Table 2), but definitive differential diagno- Described in Greek and Italian population, this IPD is sis, requires more specific tests such as platelet secretion inherited with an autosomal dominant pattern. Throm- assay, measure of granule content and flow cytometric bocytopenia ranges from mild to moderate and is usually analysis of surface receptors [26–28]. Carubbi et al.: Inherited platelet disorders diagnosis 1095 Proposed diagnostic algorithm for suspected IPDs in case of low platelet count. platelet low of IPDs in case for suspected algorithm diagnostic 1 Proposed Figure Bernard-Soulier BSS, macrothrombocyopenia; BMM, benign mediterranean synostosis; radio-ulnar with thrombocytopenia ATRUS, leukemia; myeloid AML, acute AA, acid; arachidonic leukemia; predisposition to acute disorders with platelet cytometry; FC, flow FDP/AML, familial E, epinephrine; thrombocytopenia; amegakaryocytic congenital CAMT, C, collage; syndrome; R, syndrome; QPD, Quebec disease; Willebrand von platelet-type PT-VWD, syndrome; Paris-Trousseau/Jacobsen PT/J, blood; MK, megakaryocytes; PB, peripheral syndrome; platelet gray GPS, syndrome. Wiskott-Aldrich WAS, syndrome; Velocardiofacial VCFS, 2; thrombocytopenia THC2, familial radii; absent with thrombocytopenia TAR, ristocetin; 1096 Carubbi et al.: Inherited platelet disorders diagnosis

Figure 2 Proposed diagnostic algorithm for suspected IPDs in case of normal platelet count. AA, arachidonic acid; CHS, Chediak-Higashi syndrome; E, epinephrine; FC, flow cytometry; EM, electron microscopy; GT, Glanzmann thrombastenia; HPS, Hermansky-Pudlack syndrome; PB, peripheral blood; PT-VWD, platelet-type von Willebrand disease; QPD, Quebec syndrome; R, ristocetin; SS, Scott syndrome.

Bleeding time (BT) is the time taken for bleeding to in platelet adhesion, activation, aggregation and, even- stop after an incision is made into the skin. The test is tually, formation of a plug within the aperture. The time not expensive and allows studying the entire hemostatic taken to occlude the aperture is reported as closure time process, including the role played by the vessel wall. and depends on platelet function [29]. PFA-100 is now However, its low sensitivity, specificity and poor repro- currently used in place of BT because of its better stand- ducibility, make it a suitable test only for preliminary ardization. PFA-100 is the most widely used PFA; other screening [29]. commercially available instruments are represented by In the Platelet Function Analyzer (PFA-100) test, citrate VerifyNow, Plateletworks and IMPACT-R. blood is aspirated from the sample reservoir through a Light trasmission aggregometry (LTA) tests agonist- capillary and a microscopic aperture cut into a membrane, induced changes in the turbidity (or optical density) of coated with collagen/epinephrine or collagen/ADP. The a platelet suspension while stirring the sample in a clear presence of platelet activators and high shear rates result cuvette at 37 °C. The test is commonly done using platelet Carubbi et al.: Inherited platelet disorders diagnosis 1097

Table 2 First and most common laboratory tests for the diagnosis of platelet function disorders.

Platelet Platelet Platelet aggregation count morphology

Epinephrine ADP Collagen Arachidonic Thrombin Ristocetin acid

Bernard-Soulier syndrome Decreased Large and giant Normal Normal Normal Normal Normal Absent platelets Glanzmann thromboasthenia Normal Normal Absent Absent Absent Absent Absent Absent Platelet-type von Willebrand Normal or Large platelets Normal Normal Normal Normal Normal Enhanced disease Decreased Wiskott-Aldrich syndrome Decreased Small platelets Not recommended and deficiency of δ-granules Chediak-Higashi syndrome Normal Deficiency of Impaired Impaired Impaired Impaired Impaired Impaired δ-granules Hermansky-Pudlack syndrome Normal Deficiency of Impaired Impaired Impaired Impaired Impaired Impaired δ-granules Gray platelet syndrome Decreased Large platelets Variable Variable Impaired Impaired Variable Variable Paris-Trousseau/Jacobsen Decreased Giant α-granules Not recommended syndrome Quebec syndrome Normal or Normal Impaired Normal or Normal or Normal Normal Normal decreased impaired impaired Scott syndrome Normal Normal Normal Normal Normal Normal Normal Normal Agonist PLT receptor defects Normal Normal Variable Impaired Variable Absent Variable Normal

rich plasma (PRP) and agonists used are ADP, epineph- are rarely utilized to investigate δ-granule disorders [38], rine, collagen, arachidonic acid, tromboxane analogues while are more common to assess α-granule content and ristocetin. Parameters measured include the rate of release [i.e., detection of plasma platelet factor 4 (PF4) aggregation and the maximal percentage of aggregation and β-thromboglobulin (βTG)] [39, 40]. after a fixed period of time [29, 30]. Flow cytometry (FCM) is a key technique to confirm suspected platelet disorders [28]. By using specific mono- Bernard-Soulier syndrome (BSS) clonal antibodies, it allows to detect specific glycoprotein receptor deficiencies [31, 32]. BSS is an autosomal recessive disorder with a quantita- In addition to these techniques, specific tests are tive or qualitative platelet defect due to a lack or dysfunc- available for the assessment of platelet granule number, tional von Willebrand factor (VWF) receptor, also known contents and release [33, 34]. Specifically, the α-granules as GpIb-IX-V complex, resulting in a defective adhesion of transmembrane protein P-selectin, expressed on platelets platelet to subendothelium [41]. surface once activated, is commonly detected by FCM [35]. Clinically, bleeding tendency is usually evident Transmission electron microscopy allows detect- during early childhood with easy bruising, petechiae, ing platelet granule deficiency or abnormal morphol- epistaxes and gastrointestinal bleeds. The severity of ogy based on their characteristic ultrastructure [36]. these symptoms may worsen during puberty and adult Even though electron microscopy is rarely available, it is life (i.e., menorragia). Severe bleeding usually accompa- usually required for diagnostic confirmation of specific nies hematologic stress such as surgery, trauma or tooth platelet disorders. extraction. Joint bleeds and intracranial hemorrahges are Analysis of granule content and secretion relies on rare but may occur [42]. Heterozygous patients usually the detection of proteins selectively contained in plate- present a milder bleeding tendency. let granules or expressed on the granule membrane. Laboratory diagnosis includes: detection of a vari- Among these tests, detection of ATP release using LTA able degree of thrombocytopenia (30–150 × 109/L), but combined with firefly luciferase is the first-choice method platelet count can also be normal [43]; peripheral blood to assess δ-granules secretion [37]. Radioimmunoassays smear shows increased platelet size (even in patients with (i.e., 14C-serotonin secretion from prelabeled platelets) normal platelet count), that in one third of platelets may 1098 Carubbi et al.: Inherited platelet disorders diagnosis be as large as half a red blood cell, and a few may even hematuria are less common and deep visceral hematomas be as large as a lymphocyte [44]. BT and PFA-100 are pro- are rare [50]. Bleeding symptoms occur only in homozy- longed but sensitivity of these tests is variable, depending gous patients and manifest rapidly after birth, while het- on the severity of the defect [45]. In vitro platelet aggre- erozygous condition is mostly asymptomatic since 50% of gation studies are normal with physiological agonists the normal amount of GPIIb-IIIa is at least sufficient for such as epinephrine, ADP, collagen and arachidonic acid, platelet aggregation [51]. Consequently, clinical variability except for low dose of thrombin and ristocetin that are not of this disease is high, ranging from minimal bruising to capable to induce platelet agglutination [41, 44]. severe and potentially fatal hemorrhages; anyway, gener- Platelets also fail to agglutinate when exposed to ris- ally, bleeding tendency decreases with age. tocetin and the addition of normal plasma is not effective Laboratory findings include markedly prolonged BT in rescuing this defect [44]. Confirmation of the diagno- and PFA-100 test, while platelet count and morphology sis is primarily based on FCM assessment of GpIb-IX-V are normal. LTA shows lack or severe reduction of platelet expression on platelet surface. The use of antibodies that aggregation induced by all agonists. Definitive diagno- specifically recognize CD42b reveals a severe reduction or sis relies on flow cytometric analysis of αIIbβ3 integrin complete lack of GpIbα, while other platelet antigens [i.e., expression on platelet membrane by monoclonal antibod- CD41 (GpIIb) and CD61 (GpIIIa)] are normal [46]. ies recognizing GpIIb (CD41) and GpIIIa (CD61) [52]. Differential diagnosis includes other inherited giant Based on the above mentioned laboratory findings, platelet disorders, such as May-Hegglin anomaly or Gray GT has been subclassified in three types according to the platelet syndrome (GPS), and von Willebrand disease amount of GPIIb-IIIa present per platelet, the presence (VWD). May-Hegglin anomaly is a relatively common or absence of α-granule fibrinogen and clot retraction. giant platelet disorder with clinical manifestations In type I, platelet display < 5% of the normal amount of matching with BSS; however, aggregation to thrombin surface GPIIb-IIIa and α-granule fibrinogen and clot and ristocetin is normal, while impaired response to retraction are absent. In type II, platelet GPIIa-IIIb ranges ephinephrine has been described [47]. In GPS, instead, from 10% to 20%, clot retraction is only decreased and collagen-induced aggregation is reduced and response to α-granule fibrinogen is present. The third type, known as ristocetin is normal or slightly impaired but never absent. variant thrombasthenia, is characterized by an amount of Additionally, large platelets are typically agranular at the GPIIb-IIIa equal to 50% of normal, indicating that GPIIb- blood smear [6]. VWD symptoms (see below for details) IIIa defect is qualitative rather than quantitative. may overlap with BSS and, similarly, platelet aggregation At least 126 unique mutations accounting for GT have studies reveal failure to ristocetin-induced platelet aggre- been identified in the two genes encoding for GPIIb and gation. However, the ristocetin cofactor activity measured GPIIIa: 68 in ITGAB and 58 in ITGB3, including, in order in VWD patients’ plasma and freshly washed platelets is of frequency, missense mutations (52%), nucleotide dele- uniquely reduced. Moreover, unlike BSS, VWD is not typi- tions and insertions (28%), nonsense mutations (14%), cally associated with thrombocytopenia. and alternative splicing (6%), http://sinaicentral.mssm. edu/intranet/research/glanzmann/menu. Recently, new diagnostic strategies have been pro- Glanzmann thrombastenia (GT) posed, including ELISA detection of GpIIbIIIa [53], polymerase chain reaction-single strand conformation GT is a rare autosomal recessive disease due to a deficiency polymorphism (PCR-SSCP) or allele-specific restriction or dysfunction of the platelet membrane αIIbβ3 integrin analysis for prenatal diagnosis [50]. (also known as GpIIb-IIIa) which, upon platelet activation, binds adhesive glycoproteins such as fibrinogen, VWF and fibronectin, mediating platelet aggregation [48]. In addition Platelet-type von Willebrand disease to impaired aggregation, GPIIb-IIIa deficiency accounts (PT-VWD) also for: 1) abnormal spreading of platelets on suben- dothelial matrix; 2) decreased fibrinogen content in plate- PT-VWD is a rare autosomal dominant disorder with a let α-granule; 3) reduced clot retraction; and 4) impaired quantitative and qualitative platelet defect due to a gain phosphorylation of multiple platelet proteins [49]. of function of GpIbα, a subunit of VWF receptor, on plate- GT presents as a bleeding syndrome: purpura, epix- let membrane. In PT-VWD, GpIbα displays an increased tasis, gingival hemorrhage and menorragia are almost avidity for VWF; as a consequence, platelets bind spon- constant features while gastrointestinal bleeding and taneously high molecular weight VWF multimers, with Carubbi et al.: Inherited platelet disorders diagnosis 1099 consequent platelet clumping and enhanced platelet in XLT patients. Diagnosis of WAS requires the presence clearance, causing thrombocitopenia and loss of VWF of the classical triad: microthrombocytopenia, eczema multimers [54]. and immunodeficiency. Thrombocytopenia is present at Although the enhanced platelet clumping is a risk birth, while eczema tends to develop during the first year factor for thrombosis, hemorrhagic phenotype prevails of life. Infection starts occurring early, usually in the first due to thrombocytopenia and augmented clearance of 6 months [4]. Platelet counts vary from patient to patient high molecular weight VWF multimers. Bleeding diath- – but also within individual patients – between 5 × 109/L esis is usually mild, but may become more severe after and 50 × 109/L. Mean platelet volume is between 3.5 and 5 injuries, surgery and use of antiplatelet drugs. Labora- fL, equal to half that of healthy subjects. Although many tory tests assessing platelet function show mild thrombo- reports indicate that platelet functionality in WAS and cytopenia, platelet macrocytosis and prolonged BT [55]. XLT patients is abnormal, mechanisms underlying this Platelet aggregation in response to low dose of ristocetin altered activation have not been fully elucidated yet. Addi- is enhanced, and multimeric analysis – performed by tionally, despite the potential pivotal role of WAS protein gel electrophoresis of test plasma – shows a decreased in the regulation of actin-dependent conformational amount of high molecular weight VWF multimers [56]. changes during platelet activation, in vitro data are often Plasma VWF testing shows a discordance between func- contradictory. For example, increase in F-actin content tional and non-functional test parameters, such as low and shape changes in stimulated WAS platelets appear ristocetin cofactor activity and/or collagen binding capac- to be normal [61, 62]. For these reasons, platelet function ity, compared with antigen [48]. tests are not recommended for the diagnostic work-up of Most of the clinical and laboratory features of PT-VWD WAS. are similar to VWD, due to a qualitative or quantitative defect VWF molecule. Differential diagnosis is based on ristocetin-induced aggregation. Specifically, plate- Storage pool diseases let aggregation results normal when normal platelet rich plasma (PRP) is mixed with PT-VWD plasma, while Platelets granules belong to two main categories, namely it is enhanced when PRP is added to VWD patient. Con- α- and δ-granules, containing fibrinogen, fibronectin, versely, when normal VWF is add to PT-VWD patient plate- VWF, thrombospondin, vitronectin, platelet-derived lets, aggregation results strongly enhanced, while it is growth factor (PDGF), platelet factor-IV (PF-IV), trans- normal in presence of VWD platelets [57]. However, since forming growth factor (TGF), coagulation factors and ADP aggregometry results are often ambiguous, demonstration (α-granules); ATP, serotonin, calcium, pyrophosphate of mutations in the GPIBA gene is generally necessary to (δ-granules). These factors are essential for platelet activa- provide final diagnosis. tion and aggregation, and defects in granule content and/ Recently Giannini et al. proposed also a flow cytomet- or degranulation result in disorders that may accompany ric assay to discriminate PT-VWD from VWD, quantifying both a normal or reduced platelet count. These diseases VWF-binding induced by ristocetin to fresh autologous or are known as storage pool diseases and, accordingly to to formalin-fixed donor platelets [58]. the type of granule content deficiency, are distinguished in δ-storage pool diseases (including CHS and HPS), α-storage pool diseases [including GPS, PT/JS and Quebec Wiskott-Aldrich syndrome (WAS) syndrome (QPD)] and α/δ-storage pool disease, affecting both granules [63]. WAS is a X-linked recessive disease sustained by mutation CHS is an autosomal recessive disorder with bleeding in the WAS gene coding for a protein implicated in actin tendency. Laboratory tests show a normal platelet count polymerization, and consequently required for numerous with a prolonged BT and abnormal aggregation. Addition- cellular functions such as cell structural changes, divi- ally, measurement of platelet granule components reveals sion, endomitotic process, migration and chemotaxis [59]. a reduced number of dense-granules that frequently Mutations of WAS are also described in XLT, a variant with display typically larger dimensions (giant granules). Diag- a milder clinical phenotype and more favorable progno- nosis is usually suggested by additional phenotypic trait, sis [60]. At present, more than 150 mutations in WAS gene such as oculocutaneous albinism, symptomatic neutro- have been identified. penia with recurrent infections, presence of large, perox- Mucocutaneous bleeding tendency is often the first idase-positive, cytoplasmic granules in a variety of both sign of WAS and it is the only clinically relevant symptom hematopoietic and non-hematopoietic cells [64]. 1100 Carubbi et al.: Inherited platelet disorders diagnosis

HPS is a rare autosomal recessive disease of subcel- platelets, as a consequence of the reduced or absent lular organelles present in many tissues, such as melano- α-granules content [77]. somes, lysosomes and platelet-dense granules. Deficiency Platelet aggregation studies are not helpful due of δ-granules allows including this disorder among to their variability in GPS patients. Platelet aggrega- δ-storage pool diseases. Indeed patients present with a tion induced by ADP is usually normal, but impaired bleeding diathesis that is similar to other δ-storage pool aggregation responses induced by low concentration diseases, causing easy bruising, epistaxis, menorrhagia, of thrombin or collagen have been described in same postsurgical bleeding; severity varies substantially among patients [78]. patients. BT is prolonged, and variable degrees of abnor- PT/JS is an autosomal dominant disorder character- malities can be observed by platelet function tests. When ized by mild hemorrhagic diathesis. Laboratory tests analyzed by electron microscopy, δ-granules result mark- show thrombocytopenia with normal platelet life span, edly reduced in number and characterized by peculiar increased number of bone marrow megakaryocytes with structural abnormalities that typify HPS [65]. signs of abnormal maturation and intramedullary lysis. Since this disease involves not only platelet-dense Circulating platelets display giant α-granules unable granules, but also melanosomes and lysosomes, diagnosis to release their content upon platelet stimulation with is based also on other characteristic features: tyrosinase- thrombin. These laboratory features are usually associ- positive oculocutaneous albism and ceroid-lipofuscin lys- ated with other abnormalities including mental retarda- osomal storages [66]. Ceroid-lipofuscin is a lipid-protein tion, heart defect, and facial dysmorphism [79]. complex accumulating in lysosomal organelles, which In QPD, a moderate to severe bleeding pattern is is believed to be responsible for the development of pro- associated with a gain of function in fibrinolysis. QPD gressive pulmonary fibrosis and granulomatous colitis is characterized by low levels of factor V within platelet [67–69]. To date, HPS has been associated with mutations α-granules, due to increased expression and storage of in eight human genes [65]. urokinase plasminogen activator in platelets [80]. Clinically, QPD patients present a prevalent mucocutaneous bleeding diathesis that is difficult to distinguish Gray platelet syndrome (GPS) from those of most platelet disorders, whereas other symptoms are more suggestive of a coagulation defect, In GPS the storage pool deficiency selectively hits like delayed bleeding and joint bleeds. Platelet count is α-granules. Its recessive and dominant inheritance pattern normal or moderately reduced. BT and closure time can raises the possibility that this syndrome may be caused by be normal. Platelet aggregation is absent in response to defects in more than one gene [70]. Gray platelets not only low-dose epinephrine, while normal to reduced with ADP lack the soluble molecules contained in α-granules (PF 4, and collagen. However, these aggregation findings are not β-thromboglobulin, VWF, thrombospondin, fibrinogen, QPD specific [81, 82]. fibronectin), but also the proteins that are endocytosed QPD inheritance pattern is autosomal dominant from plasma such as albumin and immunoglobulins [71]. and the genetic lesion involved is a tandem duplication The fact that α-granules membrane proteins are of PLAU, encoding for Urokinase plasminogen activa- normal [72, 73], suggests that GPS defect specifically tor. Genetic testing for PLAU duplication, using PCR and targets the process of packaging of endogenously synthe- Southern blot, has become the method of choice for defin- sized proteins in the α-granules. This packaging defect itive diagnosis of QPD. The PCR assay for QPD mutation seems to be specific for the megakaryocyte lineage, as GPS can be performed on cord blood samples to rapidly deter- patients have normal Weibel-Palade bodies in endothelial mine PLAU mutational status in a newborn from affected cells [74, 75]. parents [83]. Patients have a lifelong history of mild to severe mucocutaneous bleeding, including intracranial hemorrhages and life-threatening postsurgical bleedings. Of Scott syndrome (SS) note, some patients present a mild degree of bone marrow fibrosis [76]. SS is a rare autosomal recessive disorder affecting cal- Preliminary laboratory screening shows a prolonged cium-induced phospholipid scrambling and generation BT and thrombocytopenia, which can be as low as of thrombin on platelets. Activated Scott platelets fail to 50 × 109/L and severity may increase with time. Periph- transport phosphatidylserine (PS) from the inner to the eral blood smear demonstrates abnormally large, gray outer phospholipid leaflet of the membrane bilayer. As Carubbi et al.: Inherited platelet disorders diagnosis 1101 a result, factors Va and Xa are unable to bind the acti- in case of ADP receptor deficiency a small and rapidly vated platelet surface and, consequently, to transform reversible aggregation induced by ADP – even at high con- prothrombin into thrombin [84]. This phenotype is not centration ( ≥ 10 μM) – is seen. Additionally, other agonists restricted to platelets but can be demonstrated in other induce a defective aggregation and secretion [93]. Tests blood cells. assessing the degree of inhibition of adenylyl cyclase by Scott patients have a clinical history of provoked ADP by measuring either the platelet levels of cAMP or the bleeding episodes (menorrhagia, trauma-related hema- phosphorylation of VASP induced by prostaglandin E1, are toma, bleeding after tooth extraction, and severe postpar- recommended to confirm diagnosis [101]. tum bleeding). Thromboxane A2 receptor defects show absent aggre- Routine laboratory tests that investigate disorders of gation upon stimulation with arachidonic acid and throm- platelet function fail to reveal any deficiencies or func- boxane analogues. Response to other agonists is variable, tional aberration: BT is within the normal range, platelet with the exception for ristocetin, which is usually pre- count and morphology are normal, and no aberration of served [98, 102]. platelet aggregation, secretion, granule content have been detected [85]. Scott platelets do, however, fail to promote fibrin formation in the presence of coagulation factors. In Conclusions fact, prothrombin is not efficiently cleaved during clotting of whole blood but, unlikely coagulation factor deficien- Although in the last few years new insights in the genetic cies, this condition is not restored by addition of healthy bases and pathophysiology of IPDs have extremely donor plasma [86]. Since Scott platelets do not have improved our knowledge of these disorders, the causes altered phospholipid composition in their membrane of many of them, including the most common, are still after activation [87], flow cytometric analysis can rapidly largely unknown. Next generation sequencing strategies and effectively detect this defect through the Annexin V will hopefully help to fill this gap. binding test. The assay is based on Annexin V-binding to Many efforts are required also in order to improve and PS exposed after activation, revealing a lack of PS expo- standardize the laboratory diagnosis of IPDs. In fact, since sure after activation of Scott platelets [88]. these diseases are rare, diagnostic tests are available only A few years ago, Albrecht et al. described a specific at specialized laboratory centers. Adequate management genetic lesion associated with SS: a missense mutation in of these patients requires initially an accurate bleeding the gene coding for lipid transporter ABCA1 [89]. history and evaluation of family history (this aspect is par- ticularly relevant in children since they may have had few hemostatic challenges). Agonist platelet receptor defects The first and most common laboratory tests are represented by platelet count and peripheral blood smear. Agonist platelet receptor defects are extremely rare dis- These routinely available and relatively simple tests allow eases typified by impaired platelet aggregation in response to discriminate between bleeding disorders with normal to certain agonists, due to lack or decreased functional- platelet count from those with low platelet count, and, in ity of specific receptors. Better characterized diseases the latter group, between macro- and micro-thrombocyto- are ADP receptor and Thromboxane A2 receptor defects. penias [103]. ADP receptor defects display a heterozygous inheritance In the presence of a bleeding diathesis with a normal pattern and are due to mutations in P2Y12, accounting platelet count, platelet aggregation tests are mandatory. for a congenital deficiency or dysfunction of the receptor, An impaired aggregation to all agonists associated with leading to impaired down-stream signaling [90]. So far, pathognomonic clinical features (oculocutaneuos albi- only 13 cases have been reported in the literature [91–97]. nism, granulomatosis colitis, and pulmonary fibrosis)

Mutations involving TXA2R lead to reduced receptor suggests δ-granule defects such as CHS and HPS; electron coupling with its effector G protein and with phospholi- microscopy, although not routinely utilized in clinical pase C. An autosomal inheritance has been described for practice, leads to final diagnosis. FCM is a feasible and these diseases [98–100]. rapid technique to discriminate between GT and SS when Clinical presentation of agonist platelet receptor aggregation and clot formation are lacking [26, 27]. Aggre- defects is similar, with mild to moderate mucocutaneous gation studies are extremely informative and, generally bleeding diathesis accompanied to normal platelet count conclusive when identifying selective aggregation defects and morphology. Diagnosis relies on aggregation studies; for a specific agonist, such as epinephrine in case of QPD 1102 Carubbi et al.: Inherited platelet disorders diagnosis or ADP and arachidonic acid in case of agonist receptor Acknowledgments: This work was partially supported by defects (Figure 2). Programma di ricerca Area 1 Regione Emilia-Romagna- In the case of thrombocythopenia, bone marrow aspi- Università (ER-Università) 2010–2012 and Finanziamento rate/biopsy is generally required to investigate abnormali- Italiano per la Ricerca di Base (FIRB) RBAP10KCNS_002. ties of the megakaryocytic lineage [2]. Co-morbidities and We are grateful to Luciana Cerasuolo, Vincenzo Palermo, associated somatic defects, together with genetic testing, Domenico Manfredi and Davide Dallatana for technical lead to the final diagnosis of the great majority of inherited support. thrombocythopenias such as WAS, thrombocytopenias associated with skeletal defects, MHY9-related disorders, Conflict of interest statement XLT and PT/J (Figure 1). Among inherited macrothrombo- cythopenias, platelet function tests detain a key role in the Authors’ conflict of interest disclosure: The authors differential diagnosis between BSS and PTVWD, which stated that there are no conflicts of interest regarding the show an opposite response to ristocetin-induced aggre- publication of this article. Research support played no gation (absent and enhanced, respectively). Of note, BSS role in the study design; in the collection, analysis, and diagnosis relies also on FCM analysis of GpIb expression. interpretation of data; in the writing of the report; or in the A defective aggregation upon stimulation with collagen decision to submit the report for publication. and arachidonic acid indicates GPS (Figure 1). Research funding: None declared. Genetic testing is the final step of the diagnostic Employment or leadership: None declared. work-up of many IPDs, but, unfortunately, it is avail- Honorarium: None declared. able only for MYH9-related disorders, CAMT, X-linked thrombocytopenia, FDP/AML, BSS, PT-VWD and WAS [5] Received February 7, 2014; accepted March 11, 2014; previously (Table 1 and Figures 1 and 2). published online April 3, 2014

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Cecilia Carubbi completed her studies in Medical Biotechnolo- Elena Masselli obtained her Medical Degree in 2007 and her gies, obtaining her MSc in 2007 at the University of Parma, Italy. Hematology Board in 2012 at the University of Parma, Italy. From She is currently attending a PhD program in Biomedical Sciences March 2010 to August 2011 she was appointed as visiting assistant (University of Ferrara, Italy). Her scientific interests are represented professor at the Department of Hematology/Oncology, Mount Sinai by mechanisms of thrombopoiesis and platelet function, signaling School of Medicine, New York, USA. She is attending a PhD program in normal and malignant hematopoiesis. in Molecular Biology and Pathology at the Department of Bio- medical, Biotechnological and Translational Sciences (S.Bi.Bi.T.), University of Parma, Italy. Her research involves the molecular basis of thrombocythosis and thrombocythopenias, polycythemias and myeloproliferative neoplasms. 1106 Carubbi et al.: Inherited platelet disorders diagnosis

Biotechnological and Translational Sciences, University of Parma, Italy. Scientific interests areas: differentiation, signaling, prolifera- tion and apoptosis of blood cells.

Antonio Nouvenne graduated with full marks at the University of Parma in 2003, he took his post-graduate degree in Gastroentero logy in 2007 and his PhD in osteo-methabolic diseases and acid- base balance disorders at the same university in 2010. He currently Giuliana Gobbi, PhD, is Assistant Professor of Human Anatomy at works as hospital doctor at the Internal Medicine and Subacute the Department of Biomedical, Biotechnological and Translational Critical Care Clinic of the University Hospital of Parma. Sciences, University of Parma, Italy. She obtained her Degree in Biology in 1996 and 4 years later completed her residency in Clinical Pathology at the University of Bologna, Italy. In 2004 she obtained her Master’s Degree in Clinical and Experimental Flow Cytometry and 3 years later a PhD in Human Morphological and Molecular Sciences. She was appointed as visiting scientist at the National Human Genome Research Institute (NHGRI) Bethesda, Maryland, USA in 2000 and at the Brigham and Women Hospital (Harvard University), Boston, MA, in 2011. Her major active research interest is focused on human platelets function and megakaryocytopoiesis.

Domenico Russo, MD is Full Professor and Chair of Hematology, Department of Medical and Surgical Sciences, University of Brescia, Italy. He obtained his Medical Degree in 1982 from the University of Bologna, Italy, completed his residency in Hematology in 1986. He had a 2-year research appointment at the Laboratory of Cellular Kinetics, Hotel Dieu Hospital, University of Paris (VI), Paris, France. His scientific interests are focused on hematological diseases and stem cell transplantation.

Marco Vitale, MD is Full Professor of Human Anatomy and Chair of the Department of Biomedical, Biotechnological and Transla- tional Sciences, University of Parma, Italy. He obtained his Medical Degree in 1985 from the University of Bologna, Italy, completed his residency in Hematology in 1988 and in Biomedical Technologies 4 years later. He had a 1-year research appointment at the Jefferson Cancer Institute, Thomas Jefferson University School of Medicine Daniela Galli obtained her PhD in 2004 at the University of Rome (Philadelphia, USA). Since 2012 he has been Adjunct Professor of “La Sapienza”, Italy. From 2005 to 2007 she worked as post-doc Medicine and Member at the Institute of Human Virology at the at Pasteur Institute, Paris, France. From June 2010 she has been University of Maryland School of Medicine, Baltimore, MD, USA. His working at University of Parma, Department of Biomedical, Bio- scientific interests are focused on molecular biology and signaling technological and Translational Sciences (S.Bi.Bi.T.). Her scientific of hematopoietic cells. interests cover the studies of signal transduction in vascular and hematopoietic models.

Prisco Mirandola obtained his Degree in Biology (1992) and PhD in Molecular and Cell Biology (2001). Since 2006 he has been Associ- ate Professor of Human Anatomy at the Department of Biomedical,