Impaired secretion of platelet granules in patients with Duchenne muscular dystrophy - results of a prospective diagnostic study.

A tendency to bleed during scoliosis surgery has been reported repeatedly in Duchenne muscular dystrophy (DMD) and diagnostic studies show a prolonged bleeding time. The pathophysiological background is still not fully understood. The short dystrophin isoform dp71 is expressed in platelets and mediates contractile properties. We performed a bicentric, non-blinded, prospective diagnostic study in 53 patients with confirmed DMD. Extensive laboratory analyses included platelet aggregometry and platelet flow cytometry, as well as routine coagulation analyses. Results of laboratory diagnostics were correlated with clinical data. Patients were subgrouped and analyzed according to ambulatory status and cardiac involvement. Platelet aggregation was reduced after stimulation with ADP (adenosine triphosphate) [60%; reference range 66-84%]. In addition, in the DMD cohort the expression of platelet activation markers CD62 and CD63 (flow cytometry analyses) was significantly lower than in healthy controls, most prominent in non-ambulatory patients with cardiac involvement. There was no clear association with the location of the underlying mutations in the dystrophin gene. No further abnormalities were identified regarding primary or secondary hemostasis. This study shows that platelets of patients with DMD have decreased expression of CD62 and CD63 which are markers for platelet granule release. This may indicate that patients with DMD have an impaired platelet granule secretion which may explain to some extent the increased bleeding, especially in mucocutaneous areas and perioperatively.

Patients with Bernard-Soulier syndrome and different severity of the bleeding phenotype.

Bernard-Soulier syndrome is a rare (1:1million), hereditary bleeding disorder caused by defects of the platelet GPIb-IX-V complex. Patients suffer from mucocutaneous bleedings. Typical are thrombocytopenia, giant platelets and impaired agglutination after stimulation with ristocetin. In populations in which consanguineous marriages are common the frequency of the disorder is increased because Bernard-Soulier syndrome is mostly inherited autosomal recessively. Genetic analyses of the disease-related genes may help to gain more insights regarding the phenotype/genotype correlation. Here, we investigated several patients with Bernard-Soulier syndrome from different families. We analyzed two patients with severe bleeding symptoms from one family of middle east origin and confirmed the diagnosis by identifying a pathogenic variant in GP1BB. We compared phenotype/genotype correlation of this GP1BB mutation with the GP9 (p.Asn61Ser) European founder mutation present in 9 patients out of 4 families for whom we also performed molecular genetic analysis.

[Imaging in urinary tract infections in childhood]. / Bildgebende Diagnostik bei akuten Harnwegsinfektionen im Kindesalter.

Diagnostic strategies for extended morphological and functional clarification after symptomatic urinary tract infections in children are changing. Improved knowledge of the causes for development of renal scarring and a changing view on the importance of vesicoureteral reflux have led to a change in paradigm in recent years. The purpose of this article is to present the ongoing discussions of the causes and outcome of childhood urinary tract infections, competing diagnostic imaging methods and different diagnostic algorithms.

Cardiac thrombus with risk of fulminant pulmonary embolism in paediatric antiphospholipid syndrome.

Antiphospholipid Syndrome (APS) describes a systemic disease caused by autoantibodies to membrane components. Involving coagulation pathways, complement factors and immune cells, it results in thrombosis in any blood vessel. Its clinical presentation varies considerably depending upon the organ affected. Paediatric data on APS remain sparse. Most case reports focus on catastrophic APS with multiple small-vessel occlusions and a life-threatening course. Here, we report on a 15-year-old patient with deep vein thrombosis and a right ventricular tumour posing the risk of a fulminant pulmonary embolism. The tumour was surgically removed. Histology revealed it to be a thrombus. The patient fully recovered and is currently treated with long term anticoagulation.

Identification of acquired coagulation disorders and effects of target-controlled coagulation factor substitution on the incidence and severity of spontaneous intracranial bleeding during veno-venous ECMO therapy.

INTRODUCTION: Intracranial haemorrhage is a redoubtable complication during extracorporeal membrane oxygenation (ECMO) therapy. The underlying mechanisms of haemorrhagic diathesis are still not completely understood. This study was performed to evaluate a coagulation protocol for the regular analysis of acquired coagulation disorders and the systematic substitution of coagulation factors to reach predefined target values. We hypothesised that using this strategy would lead to the identification of acquired bleeding disorders which cannot be monitored with standard coagulation tests and that substitution of the respective factors in a target-controlled approach could have an impact on the incidence and severity of intracranial haemorrhage. METHODS: A protocol for the analysis of acquired coagulation disorders and the subsequent administration of associated factor concentrates was introduced. Previously, coagulation management was mainly based on clinical bleeding signs as the trigger for the administration of blood products. In this investigation, nineteen consecutive patients before (control group) and twenty consecutive patients after the implementation of the protocol (intervention group) have been included in the study. RESULTS: Eighty-eight percent of the patients developed factor XIII deficiency, 79% acquired von Willebrand syndrome, 40% fibrinogen deficiency and 54% of the patients showed a decline in platelet count >20% within the first 24 hours of ECMO therapy. In 6 out of 19 (31%) patients in the control group and in 2 patients out of 20 (10%) in the intervention group, intracranial haemorrhage was detected. Whilst 5 of 6 patients in the control group died because of fatal bleeding, both of the patients in the intervention group recovered with a favourable neurologic outcome. CONCLUSIONS: Veno-venous ECMO therapy leads to thrombocytopenia, factor XIII and fibrinogen deficiency as well as acquired von Willebrand syndrome. The implementation of a coagulation protocol including a standardized determination and target-controlled substitution of coagulation factors may have a beneficial impact on the incidence and severity of intracranial haemorrhage.

Congenital combined deficiency of coagulation factors VII and X--different genetic mechanisms.

Combined coagulation factor VII (FVII) and factor X (FX) deficiency (combined FVII/FX deficiency) belongs to the group of bleeding disorders in which both factors show reduced plasma activity. It may arise from coincidental inheritance of separate coagulation factor deficiencies or a common cause as large deletions comprising both gene loci. The F7 and F10 genes are located on the long arm of chromosome 13. Here, we describe 10 cases with combined FVII/FX deficiency representing both genetic mechanisms of occurrence. Genetic analyses included direct sequencing of the F7 and F10 genes and MLPA (multiplex ligation-dependent probe amplification) for detection of heterozygous large deletions. In four patients, the combined deficiency was due to a large deletion within the terminal end of chromosome 13. In the remaining six cases the deficiency resulted from coincidental inheritance of different genetic alterations affecting both genes independently. In most cases, the genetic defects were heterozygous, presenting with prolonged PT, normal aPTT and mild or no bleeding symptoms. Only in one case compound heterozygous mutations were detected in the F10, resulting in prolonged aPTT and a more severe bleeding phenotype. To avoid a misdiagnosis of combined FVII/FX deficiency, analyses of single factor activities have to be performed in all cases with prolonged PT even if aPTT is normal. Genetic analyses are substantial for correct prediction of an inheritance pattern and a proper genetic counselling.

[Therapy of inherited diseases of platelet function. Interdisciplinary S2K guideline of the Permanent Paediatric Committee of the Society of Thrombosis and Haemostasis Research (GTH e. V.)]. / Therapie hereditärer Thrombozytopathien. Interdisziplinäre S2K-Leitlinie der Ständigen Kommission Pädiatrie der Gesellschaft für Thrombose- und Hämostaseforschung e. V.

Inherited disorders of platelet function are a heterogeneous group. For optimal prevention and management of bleeding, classification and diagnosis of the underlying defect are highly recommended. An interdisciplinary guideline for a diagnostic approach has been published (AWMF # 086-003 S2K; Hämostaseologie 2014; 34: 201-212). Underlying platelet disorder, platelet count, age and clinical situation modify treatment. Exclusive transfusion of platelet concentrates may be inappropriate as potentially adverse effects can outweigh its benefit. A stepwise and individually adjusted approach for restitution and maintenance of haemostasis is recommended. Administration of antifibrinolytics is generally endorsed, but is of particular use in Quebec disease. Restricted to older children, desmopressin is favourable in storage pool disease and unclassified platelet disorders. Although licensed only for patients with Glanzmann thrombasthenia and alloantibodies, in clinical practice rFVIIa is widely used in inherited platelet disorders with severe bleeding tendency. This guideline aims at presenting the best available advice for the management of patients with inherited platelet function disorders.

[Diagnosis of inherited diseases of platelet function. Interdisciplinary S2K guideline of the Permanent Paediatric Committee of the Society of Thrombosis and Haemostasis Research (GTH e. V.)]. / Diagnose angeborener Störungen der Thrombozytenfunktion. Interdisziplinäre S2K-Leitlinie der Ständigen Kommission Pädiatrie der Gesellschaft für Thrombose- und Hämostaseforschung e. V.

Congenital disorders of platelet function are a heterogeneous group of disorders that are often not detected until bleeding occurs. In clinical settings only a few methods have proven to be useful for identification and classification of inherited platelet disorders. For a rational diagnostic approach, a stepwise algorithm is recommended. Patient history and clinical investigation are mandatory. Von Willebrand disease and other coagulation disorders should always be ruled out prior to specific platelet testing. Platelet count, size, volume (MPV) and morphology may guide further investigations. The PFA-100® CT is suited for screening for severe platelet defects. Platelet aggregometry allows assessment of multiple aspects of platelet function. Flow cytometry enables diagnosis of thrombasthenia Glanzmann, Bernard-Soulier syndrome and storage pool defects. Molecular genetics may confirm a putative diagnosis or pave the way for identifying new defects. We present an unabridged version of the interdisciplinary guideline.

Genetics of inherited platelet disorders.

The current review describes inherited platelet disorders, illustrates their clinical phenotype and molecular genetic defects. Platelets are the key molecules mediating haemostasis via adhesion, activation and clot formation at the site of injury. The inherited platelet disorders can be classified according to their platelet defects: receptor/cytoskeleton defects, secretion disorder, and signal transduction defect. Patients with inherited thrombocytopathia present with mucous membrane bleedings (epistaxis, gingival bleeding) and may present with serious life threatening bleedings following surgery or trauma. Therefore, biochemical and molecular genetic characterization of inherited platelet disorders is important to understand these disorders and to support an efficient therapy.

A prospective randomised control study: reduction of children's pain expectation using a picture book during blood withdrawal.

OBJECTIVES: Blood drawings are very painful and stressful for children. In a prospective control group study we investigated if using a picture book could reduce the children's pain expectation. In addition, the children's pain experience and the observed pain behaviour was monitored. PATIENTS/METHODS: Block-randomization were used and 120 children at the age of 6-12 years who were visiting the general pediatric and coagulation outpatient clinics were included in this study. Pain expectation and experience were assessed with the Face-Pain-Scale-Revised and the pain behavior with the Faces-Legs-Activity-Cry-Consolability Scale. Multivariate covariance analysis was used for data analysis. RESULTS: The results showed that with statistical controlling the influence of the primary pain expectation (baseline) the pain expectation before blood withdrawal was reduced significantly (p=0.001) and effectively (ES=0.56) using the picture book. Children who received no local anaesthesia reported that they felt less pain during blood drawing after reading the picture book. The few children with local anaesthesia reported no benefit from the picture book. The observed use of local anaesthesia was very heterogeneous. CONCLUSIONS: The results recommend the usage of this picture book in everyday practice, if the use of local anaesthesia could not be used in an appropriate way.

Homozygous point mutations in platelet glycoprotein ITGA2B gene as cause of Glanzmann thrombasthenia in 2 families.

Glanzmann thrombasthenia (GT) is a rare autosomal recessive bleeding disorder characterized by quantitative and/or qualitative defects of the platelet glycoprotein (GP) IIb/IIIa complex. Physiologically, the integrin GPIIb/IIIa binds Von Willebrand factor and fibrinogen on activated platelets. GT is caused by genetic alterations in ITGA2B or ITGB3 (genes encoding GPIIb and GPIIIa).This study describes 2 siblings diagnosed with GT type I associated with homozygous point mutations in ITGA2B. All patients presented with typical bleeding disorder including moderate hematomas, petechiae, and mucocutaneous bleedings.Both siblings showed severely reduced platelet aggregation especially after stimulation with collagen and adenosine diphosphate. Absence of platelet GPIIb/GPIIIa complex was determined using flow cytometry. Molecular genetic analysis revealed 2 distinct homozygous point mutations in exon 18 of ITGA2B. Family 1 was identified with c.1878G>C and family 2 with c.1787T>C substitution. While the c.1787T>C mutation causes a single amino acid substitution p.I565T, the c.1878G>C mutation (p.Q595H) is predicted to induce a mRNA splicing anomaly.These mutations were identified as cause of GT type I in the described patients. Patients with GT should be documented in a prospective register to verify the correlation between the severity of bleeding symptoms and the pathogenic mutation. This can have effects on therapeutic decisions.

Prophylactic use of clopidogrel in paediatric cardiac patients.

Thromboembolic complications in infants with congenital heart defects are common despite inhibition of platelet function with acetylsalicylic acid (ASS). Yet there is still insufficient pharmacologic data on the use of clopidogrel in infants. The adult dose of 75 mg/d is significantly higher than the dose lately recommended in infants (0.2 mg/kg/d). Moreover, we know of nonresponders to both acetylsalicylic acid and clopidogrel. Normal coagulation tests fail to identify those patients.Prospective monocentric study on 14 children (median age 5, range 0.7-84 months, 9 male, 5 female). Shunt thrombosis had occurred in 4 infants on ASS therapy. Seven days after starting clopidogrel (0.2 mg/kg/d), platelet function was tested by stimulation with ADP (4 and 10 µmol/l). We considered the range for the clopidogrel effect to be optimal if the maximum aggregation on ADP 4 µmol/l was between 30-50%.Clopidogrel 0.18-0.24 mg/kg/d in addition to ASS 2-4 mg/kg/d resulted in effective inhibition of platelet function in 93% (ADP 4 µmol/l: median 38%, range 30-63). All patients were responders. We observed neither any thromboembolic events nor severe bleeding episodes during the median 11-month follow-up period (range 1-30 mo).Testing platelet function makes clopidogrel dosing safer, and simplifies therapy adjustments in long-term treatment. A clopidogrel dose of 0.2 mg/kg/d was safe and effective in combination with ASS in this small patient cohort.

Anterior spinal artery syndrome in a 13-year-old boy 8 days after taekwondo-fight: vascular obliteration due to vessel lesion or thrombophilia?

BACKGROUND: Spinal cord infarction from anterior spinal cord syndrome (ASAS) in children is a rare pathology and comprises the following clinical symptoms: sudden onset of pain and flaccid para- or tetraparesis, bladder dysfunction, and dissociated sensory loss with impairment of pain and temperature perception. Deep sensibility is not affected. PATIENT: A 13-year-old male patient presented to our emergency department with a bilateral leg weakness. 1 week before, he had suffered a leg strain in a Taekwondo-fight from which he recovered completely. On physical examination our patient's legs were in flaccid paralysis, tone was decreased and he had dissociated sensory loss and acute retention of urine. Blood count, ESR, electrolytes, serologic tests for various pathogens and CSF examination all were normal. However, tests for values of an acute endothelial lesion were increased and he was a homozygous carrier of MTHFR-polymorphism. MRI performed on the day of admission was normal but showed dramatic changes 2 days later with increased signal intensity in the ventral aspect of the spinal cord, characteristic for an ASAS. Treatment included highdose methylprednisolone, a suprapubic bladder catheter, sufficient anticoagulation and a rapid transfer to a rehabilitation centre. DISCUSSION: We assume that a combination of the patient's prothrombotic risk factor (MTHFR-polymorphism with elevated homocysteine levels) and his trauma in the taekwondo-fight with consecutive vessel injury caused an occlusion of the artery by late emboli or a growing thrombus.