Thrombocytosis in children and adolescents-classification, diagnostic approach, and clinical management.

Secondary thrombocytosis is a frequent secondary finding in childhood infection and inflammation. Primary hereditary thrombocytosis may be caused by germline mutations within the genes encoding key regulators of thrombopoiesis, i.e., thrombopoietin (THPO) and its receptor c-MPL (MPL) or the receptor's effector kinase Januskinase2 (JAK2). Furthermore, somatic mutations in JAK2, MPL, and in the gene-encoding calreticulin (CALR) have been described to act as driver mutations within the so-called Philadelphia-negative myeloproliferative neoplasms (MPNs), namely essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF). Increasing knowledge on the molecular mechanisms and on the clinical complications of these diseases is reflected by the WHO diagnostic criteria and European LeukemiaNet (ELN) recommendations on the management of adult MPN. However, data on childhood thrombocytosis are rare, and no consensus guidelines for pediatric thrombocytosis exist. Current literature has highlighted differences in the epidemiology and molecular pathogenesis of childhood thrombocytosis as compared to adults. Furthermore, age-dependent complications and pharmacological specificities suggest that recommendations tailored to the pediatric population are necessary in clinical practice. Here we summarize literature on classification, diagnostics, and clinical management of childhood thrombocytosis.

Coexistence of antithrombin deficiency and suspected inferior vena cava atresia in an adolescent and his mother - case report and clinical implications.

BACKGROUND: Antithrombin deficiency (ATD) is an autosomal dominant thrombophilia presenting with varying phenotypes. In pediatric patients with ATD, thrombosis typically develops during the neonatal period or adolescence. However, to date there are no consistent recommendations on the therapeutic management of children with ATD. Inferior vena cava atresia (IVCA) belongs to a range of congenital or acquired vena cava malformations and is described as an independent risk factor for thrombosis. The present case report explores two cases of combined ATD and IVCA in an adolescent and his mother. CASE PRESENTATION: A 14-year-old male presented with extensive deep venous thromboses (DVTs) of both lower extremities as well as an IVCA. The patient had previously been diagnosed with an asymptomatic ATD without therapeutic consequences at that time. His mother was suffering from an ATD and had herself just been diagnosed with IVCA, too. The DVTs in the adolescent were treated by systemic anticoagulation and catheter-directed local thrombolysis causing favourable results. Yet, despite adequate oral anticoagulation the DVTs in both lower extremities reoccurred within 1 week after the patient was discharged from hospital. This time, thrombolysis could not be fully achieved. Surprisingly, probing and stenting of the IVCA was achieved, indicating an acquired IVCA which could have occurred after undetected thrombosis in early childhood. Genetic analyses showed the same mutation causing ATD in both son and mother: heterozygote missense mutation c.248 T > C, p.(Leu83Pro), within the heparin binding domain of antithrombin. This mutation was never reported in mutation databases before. CONCLUSIONS: To our knowledge this is the first case report discussing combined ATD and IVCA in two family members. Since ATDs present with clinical heterogeneity, taking a thorough family history is crucial for the anticipation of possible complications in affected children and decisions on targeted diagnostics and therapeutic interventions. Affected families must be educated on risk factors and clinical signs of thrombosis and need an immediate diagnostic workup in case of clinical symptoms. IVCA in patients with ATD could occur due to thrombotic occlusion at a very early age. Therefore, in case of family members with IVCA and ATD ultrasound screening in newborns should be considered.

Evaluation of desmopressin effect on primary haemostasis in pediatric patients with aspirin-like defect as hereditary thrombocytopathy.

OBJECTIVES: Despite about 3 decades of clinical experience with the therapy of inherited thrombocytopathies (HTP) with desmopressin (DDAVP) the mechanisms of haemostatic effects of DDAVP in these diseases remain unclear. Therefore platelet function diagnostics was carried out in whole blood (WB) from children with aspirin-like defect as one of the clinically mild forms of HTP after DDAVP administration. DESIGN AND METHODS: 11 children (age range: 3-16 years) were treated with DDAVP i.v. (0.3 µg/kg as short infusion). Before, after 120, and 240 min of DDAVP administration the following parameters were measured: platelet aggregation (PA) and ATP release induced by ADP, collagen, ristocetin and thrombin; PFA-100 closure times (CT), factor VIII activity (FVIII:C), Von Willebrand factor antigen (VWF:Ag), collagen binding activity (VWF:CB) and blood count. RESULTS: PA, ATP release and blood count were not influenced by DDAVP administration. PFA-100 CTs were markedly reduced at 120 and 240 min after DDAVP, respectively. FVIII:C, VWF:Ag and VWF:CB were increased after 120 min. CONCLUSION: The DDAVP-induced improvement of primary haemostasis in patients with aspirin-like defect is mainly due to the marked increase of the VWF. For the evaluation of the clinical effect of DDAVP administration in patients with aspirin-like defect the investigation of a larger group of patients is needed.

Therapy refractory menorrhagia as first manifestation of Hermansky-Pudlak syndrome.

INTRODUCTION: Oculocutaneous albinism (OCA) in combination with a platelet function defect caused by a disturbed release reaction from platelet δ-granules (storage pool defect - SPD) is typical for the autosomal recessive inherited Hermansky-Pudlak syndrome (HPS). CASE REPORT: A girl (age: 13 years) with OCA was hospitalized with transfusion-requiring menorrhagia. The suspicion of HPS was confirmed by results of lumi-aggregometry. Suspecting a disorder in primary haemostasis treatment with tranexamic acid (10 mg/kg body weight every 8 h i. v.), desmopressin (0.3 µg/kg body weight every 8 to 12 h) and hormonal therapy (norethisterone) was started but the menorrhagia persisted. Clinical response was finally achieved by a single injection of 100 µg/kg body weight recombinant factor VIIa (rFVIIa). CONCLUSION: The diagnosis of HPS should be suspected in patients with OCA and bleeding symptoms and is confirmed by the proof of SPD. In case of absent clinical response to desmopressin the application of rFVIIa should be considered. Hormones and antifibrinolytics are useful options in the treatment of extensive menorrhagia.

Inherited disorders of platelet function in pediatric clinical practice: a diagnostic challenge.

Hereditary disorders of platelet function are a heterogeneous group of diseases that are often complex and tend to go undetected until clinically relevant bleeding occurs. Hallmarks are epistaxis, easy bruising, mucous membrane bleeding, perioperative bleeding and menorrhagia. Bleeding may be intermittent and unpredictable. After decades of successful research on platelet biology and genetics, research findings have not been satisfactorily translated to clinical practice. The lack of robust and well- standardized test systems continues to make the diagnosis of platelet defects cumbersome for the practising clinician. Patient history and description of clinical bleeding symptoms are essential. Exclusion of von Willebrand disease, platelet count and investigation of blood smears may provide a tentative diagnosis. Light transmission aggregometry is still considered the gold standard for assessing platelet function. Due to the wide range of possible genetic defects molecular biological analyses can complement but do not substitute for other tests. The true incidence of inherited disorders of platelet function is unknown. A survey in Germany revealed that receptor-defects including Glanzmann's thrombasthenia and Bernard-Soulier syndrome and aspirin-like defects were the most frequently diagnosed platelet disorders. Of affected children 60% presented with mild and 40% with moderate to severe bleeding tendency. Epistaxis, cutaneous and mucous membrane bleeding were the most frequent symptoms. The paediatric competence network of the GTH e.V. comprises 44 collaborating centres that are caregivers to over 150 children with well-defined inherited platelet defects. A major goal of this network is to promote diagnosis of children with inherited disorders of platelet function.

[Evaluation of haemostasis in children treated with valproic acid]. / Untersuchungen zur Hämostase bei Kindern unter Valproatbehandlung.

UNLABELLED: Coagulation parameters were determined in children with valproic acid mono- and valproic acid-lamotrigin combination therapy. PATIENTS, METHODS: Monotherapy group (n = 22; mean age: 10.5 years) was compared to combination therapy (n = 7; 12.9 years) and a control group (n = 22; 8.7 years). The following parameters were measured: aggregation and ATP-release in whole blood (ADP: 20 µmol/l, collagen: 1 µg/ml, thrombin: 0.5 U/ml), PFA-100® closure times (CT), blood cell counts, global tests, VWF:Ag, VWF:CBA, factors VIII and XIII as well as fibrinogen. Bleeding symptoms were evaluated by using a questionnaire. RESULTS: For ADP- and collagen-induced aggregation as well as for ATP release no significant differences between the groups were detected. The combined therapy group showed significantly prolonged CT. Von Willebrand disease was not detected in any of the patients. The platelet count was significantly decreased in the monotherapy group. In six children a mild bleeding tendency was observed, mostly epistaxis. CONCLUSION: A clinically relevant influence of valproic acid on haemostasis was found only in few cases. However, before surgical procedures an extended coagulation diagnostics is recommended in patients with valproic acid therapy.

Platelet function in obese children and adolescents.

UNLABELLED: Platelet hyperaggregability contributes to thromboembolic events of obesity in adulthood. In obese children hyperaggregability was described in platelet rich plasma. We investigated platelet aggregation in children with obesity and lipometabolic disorders in whole blood. PATIENTS, MATERIAL, METHODS: Specimens from patients with overweight (n = 35), hypercholesterolaemia and normal weight (n = 5), overweight plus combined lipometabolic disorder (n = 5) and healthy controls (n = 20) were investigated. Aggregation and ATP release were induced by ADP (20 µmol/l), collagen (1 µg/ml) and thrombin (0.5 U/ml) using a lumiaggregometer. RESULTS: Overweight children and normal weight patients with hypercholesterolaemia exhibited no significant differences in platelet aggregation compared to controls. Contrastingly, in patients with obesity plus lipometabolic disorder the aggregation rate was significantly higher (p < 0.05) suggesting a hyperaggregable state. CONCLUSION: Obviously in obese children a hypercoagulable state exists and the slight hyperaggregability observed in whole blood in this cohort might contribute to that. Any effort should be undertaken to avoid obesity in children especially in those countries where the prevalence of obesity in childhood is continuously increasing.

Haemostatic testing prior to elective surgery in children? Not always!

In Germany, preoperative coagulation tests are commonly used, based on the belief that these tests should identify patients with an increased bleeding risk. However, published evidence does not longer support this approach for both traditional screening tests and novel techniques of global assessment of haemostasis. Unselected screening yields many false positive results and detects irrelevant disorders. It leads to postponement of surgery, anxiety in parents and patients, and is not cost effective. Even worse, it does not reliably detect relevant bleeding disorders such as the most common coagulopathy, von Willebrand disease. The bleeding history of patients and their relatives is a more effective tool to detect patients at risk. According to international guidelines and a joint statement of different German medical societies, a standardized questionnaire should be mandatory in preoperative screening. A diagnostic pathway should be employed to identify patients in whom specific tests are helpful. Because neither laboratory tests nor questionnaires can infallibly predict or exclude perioperative bleeding, guidelines for the management of these unexpected situations have to be established.

[Desmopressin testing in children with von Willebrand syndrome in haemostaseologic centers of Saxonia, Saxonia-Anhalt and Thuringia]. / Desmopressin-Tests bei Kindern mit von-Willebrand-Syndrom in den Gerinnungszentren Sachsens, Sachsen-Anhalts und Thüringens.

The influence of desmopressin on hemostasis is mediated by the release of von Willebrand factor and of coagulation factor VIII from vascular endothelium. The necessity of testing desmopressin effectiveness on hemostasis is a matter of controversy and the performance of the test is not yet standardized. For this reason the desmopressin tests in 114 children with von Willebrand syndrome (type 1, n=98; type 2A, n=12; type 2M, n=2; type 2N, n=2) carried out in 7 paediatric haemostaseologic centers were retrospectively analyzed. The effectiveness of desmopressin was assessed using defined response criteria. As expected, the test performance showed a wide variation among the centers. In 99 children desmopressin was given intravenously as a short infusion at a dosage ranging from 0.25 to 0.41 microg/kg and in 15 intranasally at an absolute dose of 40 to 300 microg. The points of time for blood taking after desmopressin application ranged from 0.5 to 12 h. The absent desmopressin response in 7 patients (6%) and the partial response in 15 indicate the necessity of testing desmopressin effectiveness before the first therapeutic use. The application of desmopressin was well tolerated by the patients.

[Renunciation of routine coagulation diagnostics before ENT operations in children. Results of a survey into the implementation of this recommendation in Dresden]. / Verzicht auf eine Routinegerinnungsdiagnostik vor HNO-Eingriffen bei Kindern. Ergebnisse einer Umfrage zur Umsetzung dieser Empfehlung in Dresden.

UNLABELLED: In Germany a recommendation was introduced by experts from several medical associations concerning the renunciation of preoperative coagulation diagnostics in ENT interventions in children with inconspicuous bleeding history and published in July 2006. In August 2007 a survey concerning the implementation of this recommendation was sent to all pediatricians and ENT doctors working in medical offices in Dresden. RESULT: The survey was answered by 23 (49%) paediatricians out of 47 who were contacted and 8 (33%) out of 24 ENT doctors. Fifteen pediatricians (65%) and 3 ENT doctors (38%) have implemented the recommendation consequently and 6 respectively 3 occasionally. Only 2 pediatricians and 2 ENT doctors did not accept the recommendation. Four paediatricians and 4 ENT doctors expressed their concerns with the implementation of recommendation. Since the implementation of this recommendation 3 children suffered from bleeding complications in ambulant ENT operations but in no case a coagulation disorder was present. CONCLUSION: The implementation of the recommendation at the regional level is practicable. Its acceptance is obviously higher in paediatricians than in ENT doctors.

[First results of the THROMKID study: a quality project for the registration of children und adolescents with hereditary platelet function defects in Germany, Austria, and Switzerland]. / Erste Ergebnisse der THROMKID-Studie: Qualitätsprojekt zur Erfassung von Kindern und Jugendlichen mit angeborenen Thrombozytenfunktionsstörungen in Deutschland, Osterreich und der Schweiz.

THROMKID is a quality project of the Paediatric Group of German Thrombosis and Haemostasis Research Society (GTH). Data from paediatric patients with hereditary thrombocytopathies (HT) treated in Germany, Austria, and Switzerland were obtained between May 2005 and August 2006. By evaluation of results of platelet function tests criteria were determined to assess the diagnosis in each patient into most likely, likely or unlikely. A total of 215 patients treated in 31 centers were identified. In 95 patients (44%) the diagnosis of HT was most likely, in 28 (13%) likely and in 92 (43%) unlikely. Taken the first two groups together (n = 123) the diagnoses were as follows: Glanzmann thrombasthenia (n = 39, 32%), Aspirin-like defect (n = 26, 21%), thrombocyte receptor defects (n = 21, 17%), storage pool disorders (n = 18, 15%), Bernard-Soulier syndrome (n = 10, 8%), Hermansky-Pudlak syndrome (n = 6, 5%) and MYH9-related hereditary makrothrombocytopenia (n = 3, 2%). The low prevalence of these diseases and the high percentage of patients with unclassified HT stresses the necessity for the establishment of a competence network for comprehensive care of these patients in the three German-speaking countries.

Congenital macrothrombocytopenia associated with a combination of functional polymorphisms in the TUBB1 gene.

Congenital thrombocytopenia in childhood and adolescence requires an extensive diagnostic workup to find the underlying reason. We report on a 13-year-old female patient who was incidentally found to have moderate thrombocytopenia which was also diagnosed in her father and brother. Within the microscopic evaluation of a peripheral blood smear macrothrombocytes were found. Immunofluorescence microscopy of the patient's platelets detected the lack of ß1-tubulin. Analysis of the TUBB1 gene revealed three known missense variants in heterozygous state which in combination might explain the ß1-tubulin defect.

Correlations between platelet aggregation, fibrinolysis, peripheral and central serotonergic measures in subhuman primates.

We have studied the relationships between whole blood and plasma serotonin (5-hydroxytryptamine, 5-HT), its major metabolite 5-hydroxyindoleacetic acid (5-HIAA) and serum lipids, platelet aggregation in the whole blood and in the platelet-rich plasma (PRP), and some fibrinolytic parameters in monkeys. Plasma 5-HT was found to be positively related to 5-HT- and ADP-induced platelet aggregation, tissue plasminogen activator (tPA) activity, serum cholesterol and LDL-cholesterol, whereas 5-HT in cerebrospinal fluid correlated inversely with serum cholesterol. Plasminogen activator inhibitor (PAI) activity was positively related to LDL. Euglobulin clot lysis time was related to both tPA and PAI activities. The significance of these findings and the possible role of 5-HT in atherogenesis and hemostasis are discussed.

Critical evaluation of the quantification of ATP release reaction in whole blood.

The influence of erythrocyte and thrombocyte content on the release of adenosine triphosphate (ATP) in whole blood was tested. Citrated blood from 39 healthy persons was diluted gradually. The release reaction was induced by arachidonic acid (1.25 mM), adenosine diphosphate (ADP; 30 microM) and collagen (1.0 and 5.0 micrograms/ml). The peak of the obtained curves was transformed into percent values of the maximal deflection by the undiluted sample (PEAK IN RELATION) and into ATP concentrations (ABSOLUTE PEAK). By rising dilution an increase of the peak in relation with all inducers was observed which was mainly due to the luminescence-optical effect. As expected the absolute peak decreased but only under arachidonic acid and collagen. Under ADP despite of the rising dilution constant amounts of ATP were released suggesting an additional release from other blood cells. High ATP release curves in response to ADP were observed in patients with pancytopenia and with thrombocytopenia when compared to health persons. ADP seems not to be suited for the measurement of ATP release reaction in whole blood. Collagen at a final concentration of 1.0 micrograms/ml was found as the optimal inducer. The ATP standard is essential for the quantification of release reaction.

N,N,N-trimethylsphingosine modifies aggregatory response and ATP release from platelets in whole blood.

We investigated the influence of N,N,N-trimethylsphingosine (TMS), a potent inhibitor of protein kinase C (PKC), on whole blood aggregation and ATP release from platelets. The preincubation with TMS at 1 microM for 2 min enhanced ATP release during arachidonic acid-induced aggregation. The ristocetin-induced agglutination was inhibited in a TMS concentration-dependent manner, which suggests that TMS may interact with the vWF receptor on platelets. TMS suppressed aggregatory response and ATP release from platelets after the addition of collagen. In contrast, the platelet aggregation induced by ADP and 12-O-tetradecanoylphorbol-13-acetate (TPA) was only slightly inhibited and the ATP release was not influenced after preincubation with TMS. Our results are in contrast to previous reported data, which were obtained using PRP and washed platelets.

Remnant-like lipoproteins stimulate whole blood platelet aggregation in vitro.

We have developed a simple, rapid assay method to measure remnant-like lipoproteins by using an immunoaffinity gel mixture of anti apo B-100 and apoA-1 antibodies to Sepharose 4B. Characterization of the unbound lipoproteins has shown that they represent chylomicron and VLDL remnant particles (RLP). Preincubation of whole blood with RLP resulted in the enhanced activation of aggregation with ADP and collagen. Such enhancement was not observed in the presence of lipoprotein deficient serum or albumin preparation. The extent of enhancement was 2.78 times by 7.5 microM of ADP and 44 times by 0.5 microgram/ml of collagen in the presence of RLP-preparation 1 (RLP-1), respectively. In the presence of RLP-2, the enhancement was 5.37 times by 7.5 microM of ADP and 102 times by 0.5 microgram/ml of collagen, respectively. On the other hand RLP slightly inhibited PRP aggregation by these agonists. Inhibitions were 19% by 7.5 microM of ADP and 18% by 1.0 microgram/of collagen in the presence of RLP-1, respectively. Incubation of whole blood with RLP did not result in the release of factors to stimulate platelets or ADP- or collagen-induced platelet aggregation in vitro. The extents of enhanced aggregation in whole blood or inhibition in PRP were not correlated with RLP-cholesterol nor RLP-protein concentrations of RLP preparations used. These results may indicate that RLP not only interact with platelets but with erythrocytes or leukocytes. Our findings support the hypothesis that the postprandial increase in remnant lipoproteins is an atherosclerotic risk factor and may be a part of the reasons of thrombotic complications by stimulating platelets in patients with remnant hyperlipoproteinemia.

In vitro effect of endothelin-1 on collagen, and ADP-induced aggregation in human whole blood and platelet rich plasma.

The effect of ET-1 on ADP- and collagen-induced platelet aggregation in whole blood and platelet rich plasma (PRP) was studied in 39 healthy volunteers. Although ET-1 itself did not cause platelet aggregation, a marked enhancement of ADP-induced aggregation after the preincubation with ET-1 for 5 min was observed in whole blood, but not in PRP. This ET-1 concentration and preincubation time-dependent phenomenon could be demonstrated only at threshold concentrations (5 and 7.5 microM) of ADP and is probably due to an interaction of ET-1 with cells which are involved in the whole blood aggregation, such as polymorphonuclear neutrophils. In whole blood and PRP an inhibition of collagen-induced aggregation after the preincubation with ET-1 was detected. In contrast to ADP, a direct influence of ET-1 on platelet activation after the addition of collagen is therefore more likely. These results suggest that human platelets may possess ET-1 receptor(s) and that ET-1 may also interact with other blood cells.

Daily variations of platelet aggregation in relation to blood and plasma serotonin in diabetes.

The circadian rhythms of platelet aggregation in the whole blood and platelet rich plasma-PRP and plasma serotonin were studied in healthy volunteers (n = 10) and diabetic patients (type II diabetes mellitus n = 12). Platelet aggregation in the whole blood induced by collagen (2 micrograms/ml), ADP (10 microM), arachidonic acid (0.5 mM) and epinephrine (10 microM), and in PRP induced by collagen (2 micrograms/ml), ADP (5 microM), arachidonic acid (250 microM), epinephrine (10 microM) and serotonin-5-HT (1 microM) was measured at 7:30, 11:30, 17:00, 23:00, 4:00 and 7:00. In healthy subjects collagen- and ADP-induced platelet aggregation in the whole blood was significantly lower at 23:00 and 4:00 when compared to values at 7:30. In PRP normal and diabetic platelet response was the lowest during the night. Diabetic platelets exhibited an enhanced response to 5-HT starting from 17:00 until 4:00 when compared to 7:30. 5-HT-induced platelet aggregation was found to be significantly higher throughout the study in DM patients over controls in parallel to plasma 5-HT. In healthy volunteers plasma 5-HT was higher at 17:00 when compared to baseline values, whereas in DM patients plasma 5-HT was elevated starting from 17:00 until 4:00. An enhanced response of diabetic platelets to 5-HT together with elevated plasma 5-HT levels may contribute, at least partly, to the pathogenesis of diabetic vasculopathy and 5HT2 receptor blockers may be of value in DM patients.

Daily variation of serum lipids in relation to the circadian rhythm of platelet aggregation in healthy male persons.

The circadian rhythm of platelet aggregation was compared with that of serum lipids in seven healthy male persons. Daily variations of remnant lipoprotein-cholesterol and of remnant lipoprotein-triglycerides were related to those of arachidonic acid-, ADP (adenosine diphosphate)-, and collagen-induced aggregation in platelet-rich plasma and to ADP-induced aggregation in whole blood, respectively. Statistical analyses indicate that the time course of remnant-cholesterol was correlated to that of ADP-induced aggregation in platelet-rich plasma and the time courses of blood cholesterol and triglyceride were correlated to arachidonic acid- and serotonin-induced platelet aggregation in platelet-rich plasma, respectively. In whole blood, the time course of remnant lipoprotein-triglyceride was correlated only to ADP-induced platelet aggregation. In contrast, the daily variation of HDL (high density lipoprotein)-cholesterol did not influence either that of platelet aggregation in platelet-rich plasma or that in whole blood. Our findings are of clinical interest regarding the development of atherosclerosis and thrombotic events in persons with an elevated level of serum lipids.

Effects of nicotine and electric footshock on peripheral serotonergic measures and on platelet aggregation in whole blood of rats.

The effects of nicotine and electric foot shock as well as of their combination on blood serotonergic measures and on whole blood aggregation have been analyzed. In rats subjected to electric footshock a rise (p < 0.05) in plasma but not in whole blood serotonin was observed, whereas this parameter was not influenced in nicotine-treated rats when compared to the control group. The combination of nicotine with electric footshock only slightly increased plasma serotonin and showed no effect on whole blood serotonin, but 5-hydroxyindole acetic acid (5-HIAA), the major metabolite of serotonin (5-HT), as well as the 5-HIAA/5-HT ratio were markedly increased (p < 0.01) suggesting an enhanced turnover of 5-HT under these conditions. The collagen-induced aggregation in whole blood was not influenced in nicotine-, in footshock- nor in combined-treated rats when compared to the controls. Our data indicate that stress as well as the combination of stress with nicotine may affect the serotonergic system which is in contrast to the exposure to nicotine alone.