Mayo Clin Proc, November 2003, Vol 78 Sebastian Platelet Syndrome 1417 extremities and slight hyperextensibility of hand and wrist joints, suggesting

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1 1416 Sebastian Platelet Syndrome Mayo Clin Proc, November 2003, Vol 78 Case Report Sebastian Platelet Syndrome: A Hereditary Macrothrombocytopenia VILMARIE RODRIGUEZ, MD; WILLIAM L. NICHOLS, MD; JON E. CHARLESWORTH, MA; AND JAMES G. WHITE, MD Sebastian platelet is a rare autosomal dominant disorder characterized by macrothrombocytopenia with granulocyte inclusions similar to those in patients with Fechtner platelet but without evidence of hereditary nephritis and sensorineural hearing loss that characterizes the latter. Although by light microscopy the granulocyte inclusions in these disorders appear morphologically similar to those found in May-Hegglin anomaly, another autosomal dominant macrothrombocytopenia, by electron microscopy the inclusions are distinct. Studies of platelet function usually suggest normal or near-normal platelet function, although mild bleeding symptoms can be associated with each of these disorders. We describe a 38- year-old woman and her 11-year-old daughter who presented with lifelong histories of mild thrombocytopenia and easy bruising. Detailed hemostatic studies showed prolonged bleeding times in the child and the mother, with the child having absent secondary wave platelet aggregation responses to epinephrine, also reflected by testing with In the differential diagnosis of hereditary thrombocytopenic disorders, giant platelet disorders (macrothrombocytopenias) are 1 group. Giant platelet s include the May-Hegglin anomaly, Fechtner platelet, Sebastian platelet, Epstein, Eckstein, Bernard-Soulier, gray platelet, Montreal platelet, Enyeart anomaly, and patients with enlarged platelets but no other characteristic features (Table ). Giant platelets also have been reported in association with the velocardiofacial, with platelet glycoprotein Ib abnormalities and mitral valve insufficiency, and with platelet glycoprotein IV abnormalities Mediterranean macrothrombocytopenia is a disorder characterized by thrombocytopenia and large platelets with no bleeding symptoms. Its pathogenesis is unclear. 15 Sebastian platelet, described first by Greinacher et al, 16 is characterized by autosomal dominant the platelet function analyzer (PFA-100 device). The mother s hemostatic studies were normal including platelet aggregometry, PFA-100 testing, and platelet flow cytometry. By light microscopy the blood smears of both individuals showed neutrophil inclusions, and their platelets were mildly enlarged but were not giant. Electron microscopy showed the neutrophil inclusions seen in classic Sebastian platelet or Fechtner platelet. These 2 cases expand the description of Sebastian platelet to include individuals with large but not giant platelets and mild or minimal thrombocytopenia. The differential diagnosis of hereditary thrombocytopenias is reviewed briefly. Mayo Clin Proc. 2003;78: ADP = adenosine diphosphate; DDAVP = desmopressin; MYH9 = nonmuscle myosin heavy chain IIA gene; PFA-100 = platelet function analyzer; PMN = polymorphonuclear thrombocytopenia with giant platelets and granulocyte inclusions similar to those seen in Fechtner platelet but without the nephritis and sensorineural hearing loss that occur in the latter. The granulocyte inclusions in the Sebastian platelet and Fechtner platelet are distinguished ultrastructurally from those in the May- Hegglin anomaly in that they are more spherical and do not contain longitudinally arranged filaments. 1,17 Platelet aggregation responses to various agonists are usually normal or near normal in all 3 disorders. A variant of Sebastian platelet was described recently in which the granulocyte inclusions are ultrastructurally distinct compared with the classic Sebastian platelet. 1 We present clinical and laboratory features of 2 new patients with classic Sebastian platelet but with only mild thrombocytopenia and mildly enlarged platelets; also we discuss the differential diagnosis of hereditary thrombocytopenia. From the Division of Pediatric Hematology/Oncology (V.R.), Division of Hematology and Internal Medicine, Special Coagulation Laboratory (W.L.N.), and Department of Biochemistry and Molecular Biology, Mayo Core Electron Microscopy Laboratory (J.E.C.), Mayo Clinic, Rochester, Minn; and Department of Laboratory Medicine and Pathology and Department of Pediatrics, University of Minnesota, Minneapolis (J.G.W.). Address reprint requests and correspondence to William L. Nichols, MD, Special Coagulation Laboratory, Mayo Clinic, 200 First St SW, Rochester, MN ( nichols.william@mayo.edu). REPORT OF CASES Case 1 An 11-year-old white girl presented with a history of lifelong easy bruising. A detailed review of the hemostatic history suggested no other types of abnormal bleeding; however, the patient had no prior hemostatic challenges such as surgery or dental extractions. Initial physical examination disclosed a few small bruises on the lower Mayo Clin Proc. 2003;78: Mayo Foundation for Medical Education and Research

2 Mayo Clin Proc, November 2003, Vol 78 Sebastian Platelet Syndrome 1417 extremities and slight hyperextensibility of hand and wrist joints, suggesting a mild structural collagen defect that contributed to her mild bleeding symptoms. Laboratory evaluation showed mild thrombocytopenia (reference ranges shown parenthetically), with a platelet count of /L ( /L) with mildly enlarged platelets. The mean platelet volume ranged from 14 to 17 fl (7-11 fl) on 5 peripheral blood cell count examinations, and the platelet count averaged /L. Peripheral blood smear examination confirmed mild thrombocytopenia and mildly enlarged platelets and revealed light blue staining inclusions in neutrophils. Hemostatic testing showed a markedly prolonged Ivy bleeding time (Surgicutt device, International Technidyne, Edison, NJ) on 2 occasions (>15 minutes; normal is 8 minutes). Platelet aggregation responses to all agonists were normal, except for absent second wave responses after epinephrine stimulation. Platelet function analyzer (PFA-100, Dade Behring, Miami, Fla) closure times ( in vitro bleeding times ) were prolonged abnormally for epinephrine-collagen coated cartridges (>300 seconds, normal 165 seconds) but were normal for adenosine diphosphate (ADP)- collagen coated cartridges. Levels of factor VIII and von Willebrand factor were normal as were prothrombin time, activated partial thromboplastin time, thrombin time, and factor XIII screening assay. At the times of platelet function testing, the patient was taking methylphenidate for management of an attention deficit hyperactivity disorder and had taken amoxicillin on 1 occasion. Before elective tonsillectomy, desmopressin (DDAVP) infusion shortened the patient s bleeding time from more than 15 minutes to 12 minutes when tested 1 hour after the infusion. Several months later she underwent a tonsillectomy, performed to manage nocturnal breathing problems. Preoperative DDAVP was infused (0.3 µg/kg body weight) followed by a second infusion 48 hours later. ε-aminocaproic acid was given orally for 7 days (3 g every 6 hours). Ten days after surgery, the patient developed postoperative oropharyngeal bleeding; DDAVP was infused, followed by another course of ε-aminocaproic acid for 7 days. No surgical intervention or transfusion of blood products was required. Several months after tonsillectomy, the patient underwent umbilical and inguinal herniorrhaphy. Preoperatively, she received DDAVP intravenously (0.3 µg/kg per dose); postoperatively, she received ε-aminocaproic acid orally for 7 days. She had no intraoperative or postoperative bleeding complications. Case 2 A 38-year-old white woman, the mother of case 1, was known to have lifelong thrombocytopenia (platelet range, /L to /L; mean, 8 observations) and mild Table 1. Hereditary Thrombocytopenias With Large Platelets 1-15 Disorder Features May-Hegglin Autosomal dominant, normal or near-normal anomaly platelet function, granulocyte inclusions Sebastian platelet Autosomal dominant, normal or near-normal syndome platelet function, granulocyte inclusions Fechtner platelet Autosomal dominant, normal or near-normal platelet function, granulocyte inclusions, nephritis, deafness, cataracts Epstein Autosomal dominant, nephritis and deafness, platelet hypofunction, no granulocyte inclusions Eckstein Like Epstein except platelet function normal Bernard-Soulier Autosomal recessive, absent platelet glycoprotein Ib/IX, no aggregation response to ristocetin, moderately severe platelet dysfunction Gray platelet Autosomal inheritance, absence of platelet α- granules, absent granulomere (gray platelets), mild to moderate platelet dysfunction Montreal platelet Autosomal dominant, prolonged bleeding time, spontaneous platelet aggregation Enyeart anomaly Autosomal inheritance, mild to severe bleeding, small inclusion bodies in platelets Giant platelets with Autosomal recessive, defective platelet glycoprovelocardiofacial tein Ib (22q11.2), normal platelet aggregation, absent bleeding symptoms, conotruncal heart defects, learning disability Giant platelets with Autosomal recessive, decreased platelet abnormal mem- aggregation responses to adenosine brane glycoproteins diphosphate, thrombin, arachidonate, absent and mitral valve glycoprotein Ia, Ic, IIa, mild bleeding insufficiency Familial macro- Autosomal dominant, glycoprotein IV thrombocytopenia abnormality, platelet aggregation to adenosine with glycoprotein diphosphate and epinephrine slightly IV abnormality decreased, no neutrophil inclusions, variable degree of bleeding Mediterranean Pathogenesis unclear, no inclusion bodies, no macrothrombo- bleeding cytopenia platelet enlargement (mean platelet volume, 13.7 fl; range, fl). The patient had a history of menorrhagia that improved after taking oral contraceptives. She had 2 pregnancies and deliveries with no hemostatic complications and had undergone several major surgical procedures, including exploratory laparotomy and bilateral breast reduction surgery, with no associated bleeding complications. At the time of platelet function testing, the patient was taking an oral contraceptive and amoxicillin. Peripheral blood smear examination showed basophilic neutrophil inclusions and mild thrombocytopenia with mildly enlarged platelets. Hemostatic evaluation showed a prolonged bleeding time (>15 minutes). Platelet aggregation responses to arachidonate, ADP, epinephrine, collagen, and ristocetin agonists were normal. Prothrombin time, activated partial thromboplastin time, thrombin time, factor VIII, and von Willebrand factor (von Willebrand factor antigen and ristocetin cofactor activity) were normal, as was the factor

3 1418 Sebastian Platelet Syndrome Mayo Clin Proc, November 2003, Vol 78 were otherwise morphologically normal, including dense granule content assessed by whole mount examination of platelet-rich plasma and thin sections of pelleted platelets. 1,18,19 Electron microscopy of the blood buffy coat of both patients confirmed the granulocyte inclusions and revealed their fine structure (Figures 1 and 2). The inclusions were irregular but relatively circular. The matrix contained clusters of ribosomes and a few unorganized filaments in an otherwise structureless matrix. No rough endoplasmic reticulum was observed in the leukocyte inclusion bodies, and the clusters of ribosomes were not membrane-enclosed. Figure 1. Case 1. Thin-sectioned blood buffy coat showing a granulocyte with nuclear lobe (N) and inclusions (arrows) that have ultrastructural features typical of classic Sebastian (uranyl acetate and lead citrate, original magnification 17,000). The relatively structureless matrices of the inclusions contain clusters of ribosomes and a few unorganized filaments. XIII screening assay. Flow cytometry of platelets in diluted whole blood revealed normal glycoprotein IIb-IIIa activation and α-granule P-selectin response to stimulation with 10 nmol/l human α-thrombin or 100 µmol/l ADP. 18 ELECTRON MICROSCOPY Electron microscopy of blood platelets from both patients reconfirmed the presence of mildly enlarged platelets that Figure 2. Case 2. Thin-sectioned blood buffy coat showing nucleus (N) and granulocyte inclusions (arrows) that are morphologically consistent with classic Sebastian (uranyl acetate and lead citrate, original magnification 17,000). DISCUSSION Three forms of hereditary thrombocytopenia with giant platelets and inclusion bodies in polymorphonuclear (PMN) leukocytes have been described: the May-Hegglin anomaly, the Sebastian platelet, and the Fechtner platelet. The May-Hegglin anomaly is the most prevalent of these uncommon disorders and is characterized by autosomal dominant inheritance of thrombocytopenia with giant or large platelets and spindle-shaped inclusion bodies in the PMN leukocytes. 2,20,21 Thrombocytopenia is variable, usually between and platelets/ml. Because of the giant platelets, accurate platelet enumeration is frequently difficult, especially with automated cell counting instruments; manual (microscopic) counting may be necessary, such as indirect counting by determining the ratio of platelets to erythrocytes by microscopy of the blood smear and deriving the estimated platelet count from the automated erythrocyte count. Platelet function is often normal but can be mildly decreased; however, it is sometimes difficult to assess because of thrombocytopenia. Some patients experience a bleeding tendency, whereas many are asymptomatic. May-Hegglin inclusions are large and spindle-shaped and contain parallel filaments studded with clusters of ribosomes. 1,21 Fechtner platelet was described in 1985 and is characterized by autosomal dominant nephritis, sensorineural hearing loss, cataracts, and macrothrombocytopenia with PMN inclusions. 18,22,23 The granulocytes have spindle-shaped inclusions, visible by light microscopy as light blue with Wright or Giemsa staining. By electron microscopy, the inclusions are relatively spherical and contain a few unorganized filaments in an otherwise clear matrix devoid of granules or other organelles. The Sebastian platelet was described in 1990 by Greinacher et al. 16 This thrombocytopenic disorder shows the same platelet and leukocyte morphology observed in the Fechtner platelet. The distinguishing feature is the absence of the clinical findings of Fechtner platelet that are similar to those in

4 Mayo Clin Proc, November 2003, Vol 78 Sebastian Platelet Syndrome 1419 Alport (autosomal dominant nephritis, sensorineural deafness, and cataracts). Bleeding manifestations are variable in the Sebastian platelet and Fechtner platelet ; some patients show a mild bleeding tendency or no bleeding manifestations, whereas others present with postoperative hemorrhage ,22,23 Recently, a new morphologic variant of Sebastian platelet was identified. 1 Other macrothrombocytopenic disorders include Epstein, Eckstein, Enyeart anomaly, and Montreal platelet. 4-6 Epstein is characterized by autosomal dominant interstitial or mixed nephritis associated with sensorineural deafness. Affected individuals have macrothrombocytopenia, a prolonged bleeding time, defective platelet adhesion, and abnormal aggregation in response to collagen and epinephrine; however, they reportedly have no granulocyte inclusions characteristic of Fechtner platelet. Identification by light microscopy or electron microscopy of granulocyte inclusions in Fechtner platelet, Sebastian platelet, and May-Hegglin anomaly may be challenging, and the inclusions may escape detection because of staining quality, the microscopist s lack of expertise, or the instability of the inclusions after blood sampling. 2,16 Eckstein has the same clinical features as Epstein except for normal platelet function. 4-6 Enyeart anomaly is characterized by giant platelets with inclusion bodies, thrombocytopenia, and mild to severe bleeding problems but no leukocyte inclusions. 12 The Montreal platelet is a giant platelet disorder with spontaneous platelet aggregation but no leukocyte inclusions. 4,8 These 3 s are extremely rare, and reported cases may represent unique individuals or families. 4-6,8 Tables 1 and 2 provide an outline of diagnostic features of hereditary thrombocytopenias, organized according to platelet size: macrothrombocytopenias in Table 1 and hereditary thrombocytopenias without large platelets, including disorders with normal platelet size or small platelets, in Table Of note, other hereditary thrombocytopenic disorders may lack the phenotypic or syndromic features classified in Tables 1 and 2, presenting only as autosomal dominant, autosomal recessive, or X-linked thrombocytopenias with variable platelet count, size, and function. 7 The genetic defects of the giant platelet s (May-Hegglin anomaly, Fechtner platelet, Sebastian platelet, and Epstein ) have been mapped to chromosome 22q This region contains a gene that encodes the nonmuscle myosin heavy chain IIA (MYH9) The MYH9 gene product accounts for approximately 2% of platelet protein content. Different Disorder Type 2B von Willebrand disease Platelet type (pseudo) von Willebrand disease Autosomal dominant, VWF mutation with increased binding affinity for blood platelets (glycoprotein Ib/IX), increased sensitivity to low-dose ristocetin-induced platelet aggregation, low VWF ristocetin cofactor activity, absence of large VWF multimers Autosomal dominant, platelets with increased binding affinity (glycoprotein Ib/IX mutation) for plasma VWF, increased sensitivity to lowdose ristocetin-induced platelet aggregation, low VWF ristocetin cofactor activity, absence of large VWF multimers X chromosome linked recessive, tiny platelets, immunodeficiency, frequent bacterial infec- tions, eczema, normal or near-normal platelet function Autosomal recessive, bilateral absence of radii, decreased megakaryocytes in bone marrow, platelet hypofunction in some patients Autosomal dominant or recessive, or X chromo- some linked, normal or abnormal platelet function, syndromic or nonsyndromic Wiskott-Aldrich (Campbell) Thrombocytopenia absent radius (TAR) Other thrombocytopenias with normalsized or small platelets Table 2. Hereditary Thrombocytopenias Without Large Platelets* 3-8 *VWF = von Willebrand factor. Features genetic alterations in this gene have been described in individuals with May-Hegglin anomaly, Sebastian platelet, Fechtner platelet, and Epstein The identified mutations in MYH9 are in highly conserved and critical positions in both the globular head and the coiled-coil domains that are expected to result in altered assembly and stability of myosin, thus potentially affecting platelet and leukocyte structure and function The identified molecular defects might help explain the pathophysiology of these disorders and their potential diagnostic utility Immunofluorescent or immunocytochemical studies to detect neutrophil nonmuscle myosin heavy chain-a in neutrophil inclusion bodies are potentially useful screening or diagnostic tests for macrothrombocytopenic disorders with or without obvious inclusion bodies. 33 Abnormal subcellular localization of neutrophil nonmuscle myosin heavy chain-a has been described in neutrophils of affected individuals with MYH9 mutations. 33 Our 2 cases represent the classic Sebastian platelet. 1 Clinically, both patients lacked the findings of hearing loss, ocular abnormalities, and nephritis present in Fechtner platelet. Their platelets were enlarged but not giant (equivalent to or larger than the size of erythrocytes), in contrast to previous reports. 1,16,17 Electron microscopy showed that the granulocyte inclusion bodies have a structure characteristic of either the classic Sebastian platelet or the Fechtner platelet but inconsistent with the May-Hegglin anomaly.

5 1420 Sebastian Platelet Syndrome Mayo Clin Proc, November 2003, Vol 78 Case 2 had no history of bleeding complications after major surgical challenges. Hemostatic evaluation showed normal findings, including extensive platelet function testing that was normal except for a prolonged bleeding time. However, case 1 had a prolonged bleeding time that shortened but did not correct after DDAVP infusion. Platelet aggregometry showed an absent second wave response to epinephrine stimulation. This was reflected by prolongation of the epinephrine-collagen cartridge closure time using the PFA-100 assay system. The possible influence of methylphenidate effects on platelet function could not be excluded. No other hemostatic laboratory abnormalities were identified. This patient may have had a mild collagen structural defect that could have contributed to the prolonged bleeding time and posttonsillectomy bleeding. Several disorders should be remembered when evaluating a patient with thrombocytopenia. Differential diagnosis of thrombocytopenic disorders includes 2 groups: acquired and hereditary. The acquired thrombocytopenic disorders include abnormalities in platelet distribution (eg, splenomegaly), reduced platelet production due to megakaryocytic hypoplasia or bone marrow malignancy, and increased destruction or consumption that may be immune-mediated (eg, autoimmune thrombocytopenia) or nonimmune in origin (eg, disseminated intravascular coagulation and fibrinolysis). Peripheral blood smear evaluation is necessary to confirm thrombocytopenia, to assess the size and morphology of the platelets, and to evaluate whether the leukocytes contain inclusions, thereby providing clues for differentiating between acquired and hereditary thrombocytopenias. If either large platelets or inclusions in leukocytes are observed on a peripheral blood smear, electron microscopy can help establish a diagnosis. Hereditary thrombocytopenia can be misdiagnosed as an autoimmune thrombocytopenia, and unnecessary treatments such as corticosteroids, immunoglobulin infusions, or splenectomy may be given. For evaluation of thrombocytopenic patients, either children or adults, it is imperative that a careful history, physical examination, and blood smear examination are undertaken to narrow the differential diagnosis. Finally, electron microscopy of peripheral blood platelets and leukocytes can provide detailed ultrastructural analysis to make a diagnosis in some of these thrombocytopenic s, especially in disorders in which inclusion bodies are seen in the granulocytes on peripheral blood smear examination by light microscopy. We thank Randall S. Miller (Mayo Clinic Hematology Coagulation Research, WG Owen Laboratory) for performing the platelet flow cytometric studies. REFERENCES 1. White JG, Mattson JC, Nichols WL, Luban NL, Greinacher A. 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