Anti-adalimumab antibodies in juvenile idiopathic arthritis: frequent association with loss of response.

OBJECTIVES: We aimed to determine how loss of response (LOR) to adalimumab (ADA) in juvenile idiopathic arthritis (JIA) may be related to anti-ADA antibodies (AAA). METHOD: AAA and ADA levels were measured in 23 consecutive patients with JIA responding significantly to treatment with ADA. RESULTS: Six out of 23 (26%) patients developed AAA and had low ADA levels. Five out of six AAA-positive patients experienced LOR. In these patients use of concomitant methotrexate (MTX) was significantly lower. CONCLUSIONS: The occurrence of AAA is a frequent event associated with LOR. Monitoring of AAA and serum ADA levels should be considered in JIA patients under ADA therapy.

Normalization of haemostatic alterations in overweight children with weight loss due to lifestyle intervention.

BACKGROUND: Obesity has been shown to be associated with a hypercoagulable state. However, the effect of weight loss on these haemostatic alterations has not been studied yet with an overall function test such as the thrombin generating test (TG) in obese children. METHODS: We prospectively determined weight status as SDS-BMI, fibrinogen, and performed TG determining time to peak (TTPeak), peak, time preceding the thrombin burst (lag-time), and "endogenous" thrombin potential (ETP) in 27 overweight children (mean age 11.9 ± 2.4 years, 45% female, mean BMI 27.5 ± 5.6 kg/m(2), mean SDS-BMI 2.31 ± 0.48) both at baseline and after 1 year lifestyle intervention based on nutrition education, exercise therapy, and psychological care. Furthermore, thrombin generating test and fibrinogen were determined in 50 healthy children of same age. RESULTS: Overweight children demonstrated significantly higher fibrinogen levels (p = 0.013), shorter lag-time (p < 0.001), and TTPeak (p = 0.028) compared to normal-weight children. ETP (p < 0.001) and peak (p < 0.001) were significantly higher in overweight than in normal-weight children. The overweight children reduced their degree of overweight significantly (-0.4 5 ± 0.22 SDS-BMI; p < 0.001). At the end of the lifestyle intervention, all haemostatic alterations normalized (significant decrease of fibrinogen (p = 0.036), ETP (p = 0.034), and peak (p = 0.001); significant increase of lag-time (p = 0.040) and TTPeak (p < 0.001)). CONCLUSIONS: The alterations in the haemostatic system in obese children normalized after weight loss due to lifestyle intervention demonstrating their reversibility.

[Is the DDAVP-test helpful to diagnose children?]. / Trägt ein DDAVP-Test bei Kindern zur Diagnose bei?

UNLABELLED: It is very difficult to determine if patients with a moderate low level of VWF parameters have mild disease or if they are just low normal (so called grey area of VWD). This applies particularly to pediatrics, because it is difficult to evaluate the bleeding history of children. Al our centres every child diagnosed with vWD gets DDAVP to test the response for it. This study was done to evaluate the DDAVP- test as a diagnostic tool. PATIENTS, METHODS: A retrospective analysis of data obtained with routine DDAVP administration for test purposes in 52 patients with borderline von Willebrand disease at the haemophilia centre Graz was done. The increase of VWF:Ag, VWF:RiCof and FVIII:C has been document and compared. RESULTS: All of our patients had a very good response after application of DDAVP. The increase of VWF:Ag, VWF:RiCof and FVIII:C was compared in patients with positive and negative bleeding anamneses. The patients with positive anamneses had significantly lower parameters at the beginning. The increase of VWF parameters did not differ significantly between the groups at the different time-points. These results demonstrate that a positive anamnesis is not significantly associated with a lower increase. On the other side a high increase is not associated with a negative anamnesis. CONCLUSION: It is not possible to use the DDAVP test as a diagnostic tool for patients within the diagnostic grey area of VWD.

High microparticle concentration in cord plasma.

UNLABELLED: We investigated if differences in the microparticle concentration and activity between newborn cord plasma and adult plasma exist. METHODS: To enumerate and characterize microparticles (MP) FACS and ELISA were used.The effect of microparticles derived tissue factor (TF) on thrombin generation was measured indirectly by CAT (calibrated automated thrombography). RESULTS: The flow cytometric measurements revealed an increased microparticle concentration in newborn cord compared with adult plasma. By the use of ELISA a significantly increased procoagulant activity of microparticles was found in newborn cord plasma as compared to adult plasma. Initiation of thrombin generation by adding phospholipids alone resulted in a significant lower prolongation of the lag time, time to peak in cord plasma, while the decrease of endogenous thrombin potential (ETP) and peak was comparable between newborns and adults. CONCLUSION: Our results show a higher impact of microparticles on the haemostatic system of newborns than on that of adults. The three methods suggest a somewhat increased microparticle activity in newborn cord plasma, but argue against strong platelet activation during birth.

Comparative evaluation of PAR1, GPIb-IX-V, and integrin αIIbß3 levels in cord and adult platelets.

BACKGROUND: Newborn platelets show hyposensitivity in vitro to many important agonists. However, sensitivity of platelets to these agonists is crucial for a functional clot formation. Nevertheless newborns have an excellent hemostasis. Hence, we examined levels of PAR1 thrombin receptor, GPIb-IX-V (CD42b), and Integrin αIIbß3 in newborn and adult platelets using Western blot analysis. Materials, methods: Platelets of adult and cord blood were isolated, washed, and lysed. Protein samples were separated by SDS-PAGE and blotted on nitrocellulose membranes. Receptors were visualized using immunodetection and evaluated densitometrically. Statistical analysis was performed using SPSS 16.0. RESULTS: We found significantly lower levels of PAR1-receptors and higher levels of CD42b in newborn platelets as compared to adult platelets. Levels of Integrin αIIbß3 in newborn platelets were comparable to adult platelets. CONCLUSION: A lower content of PAR1-receptors explains very well the hyposensitivity of cord platelets to thrombin. Higher levels of CD42b may additionally support the effect of larger more adhesive multimeric vWF in newborn plasma.

Relationship between thrombin generation and carotid intima-media thickness.

UNLABELLED: Increasing evidence indicates that thrombin plays a role not only in thrombosis but also in the progression of atherosclerosis. AIM: The relationship between thrombin generation and intima-media thickness (IMT) as an index of subclinical atherosclerosis was investigated. Participants, material, methods: We examined 163 asymptomatic middle-aged persons free of overt clinical atherosclerotic disease. They underwent ultrasonography of the common carotid arteries. In addition, thrombin generation was measured by means of CAT (calibrated automated thrombography). For our study we divided the healthy study participants into three age groups (<45, 45-60 and >60 years). RESULTS: A significant positive correlation was seen between endogenous thrombin potential (ETP) (p = 0.012), time to peak (TTP) (p = 0.033) start tail (p = 0.007) and carotid IMT in the group of healthy volunteers younger than 45 years. CONCLUSION: We demonstrated that in adults younger than 45 years without clinically overt atherosclerotic disease ETP was significantly associated with carotid IMT. It is tempting to speculate that ETP may serve as an index for subclinical atherosclerosis in persons below 45 years.

Newborn platelets: lower levels of protease-activated receptors cause hypoaggregability to thrombin.

Newborn platelets show in vitro hypoaggregability to thrombin. Sensitivity of platelets to such a potent agonist is crucial for a functional clot formation. Nevertheless, newborns have an excellent hemostasis. We wanted to investigate the reason for this impairment by comparatively analysing levels of receptors known to be involved in thrombin signaling in newborn and adult platelets. Platelets of adult and cord blood were isolated, washed, and lysed. Resulting protein samples were separated by SDS-PAGE and blotted on nitrocellulose membranes. Receptors were visualized using immunodetection and evaluated densitometrically. Thrombin receptor activating peptide induced platelet aggregation was measured in citrated whole blood on a Multiplate analyzer. Statistical analysis was performed using SPSS 16.0. Significantly lower levels of protease-activated receptors (PAR1, PAR4) and higher levels of glycoprotein Ibα (GPIbα) were found in newborn platelets as compared to adult platelets. Platelet aggregation was lower in newborn samples than in adult controls and values correlated with the corresponding PAR levels. Our results suggest that lower levels of protease-activated receptors contribute to the poor thrombin induced aggregation observed with newborn platelets, which can not be compensated by higher levels of GPIbα.

Haemostatic alterations in overweight children: associations between metabolic syndrome, thrombin generation, and fibrinogen levels.

BACKGROUND: The metabolic syndrome (MetS) is associated with central obesity and leads to increased morbidity and mortality due to cardiovascular disease (CVD). Since obesity is associated with a hypercoagulable state, it has been speculated that hypercoagulation is linking MetS to CVD. METHODS: We prospectively examined 81 overweight children and 32 normal-weight children aged 10-16 years. We analyzed blood pressure, fasting lipids, glucose, insulin, fibrinogen, and thrombin generating test determining time to peak (TTPeak), peak, time preceding the thrombin burst (lag-time), and 'endogenous' thrombin potential (ETP). RESULTS: Overweight children demonstrated significantly higher fibrinogen levels (p<0.001), shorter lag-time (p<0.001), and TTPeak (p=0.038) compared to normal-weight children. Furthermore, ETP (p<0.001) and peak (p<0.001) were significantly higher in overweight than in normal-weight children. Fibrinogen and all parameters of the clotting test correlated significantly (p always <0.05) to body mass index (BMI) but not significantly to insulin resistance index HOMA-IR or occurrence of MetS in multiple linear backward regression analyses adjusted for age and gender. CONCLUSIONS: The increased fibrinogen levels and the changes in the thrombin generation test points towards a haemostatic alteration in overweight children. The parameters of the clotting test were related to the degree of overweight but not to insulin resistance or occurrence of MetS questioning a direct association between MetS and the coagulation system. Longitudinal studies are needed to confirm these findings.

Diagnostic and therapeutic considerations on inherited platelet disorders in neonates and children.

Inherited disorders of platelets constitute a group of rare diseases that give rise to bleeding syndromes of variety severity, with more severe cases being first diagnosed during infancy and childhood. To appropriate diagnose a platelet function disorder during early childhood the knowledge of the physiological characteristics of platelets in the paediatric population is mandatory. Apart from thrombocytopenia which is quite common in neonates and children the present overview is aimed to focus on inherited platelet function disorders. Furthermore, knowledge on platelet maturation and reference values according to age are given, and a diagnostic strategy specifically adapted to a pediatric population is presented on the bases of plasmatic and molecular laboratory methodologies. Finally, therapeutic approaches are briefly summarized (antifibrinolytic agents, Desmopressin, HLA-matched platelets, recombinant factor VIIa).

Malignant stroke in an adolescent with a homozygous MTHFR 677CT mutation and intake of hormonal contraceptives.

UNLABELLED: Severe stroke in children and adolescents with its devastating long term consequences remains a rare disorder with many open questions. Beside the well known risk factors such as infection or congenital and acquired heart disease, coagulation disorders have to be considered in the differential work up of the underlying aetiology. Here we report the case of an adolescent with a homozygote MTHFR 677CT mutation suffering a malignant stroke shortly after the start of oral contraceptives. CONCLUSION: Since prevention seems easily feasible, general screening for MTHFR mutations might be worthwhile in women before starting oral contraceptives.

[Austrian haemophilia registry: up-date 2008 ]. / Osterreichisches Hämophilie Register: Update 2008.

The treatment of haemophilia requires continuous development of knowledge related to various aspects of diagnosis and therapy. It is, therefore, essential to collect valid and representative data, which are comparable on an international level. The Austrian Haemophilia Registry was set up by the Scientific Advisory Panel of the Austrian Haemophilia Society and by the patient organisation. For the design, it was decided to divide the registry into three sections, two concerning quality control and a third concerning scientific questions, the latter requiring written informed consent. A web-based software is used to collect data. Transfer and storage of data are secured and the server is situated in a computer center with video and access control. Data entry was initiated early 2008. Currently, only preliminary data are available. Our further focus is on continued data entry, which will further enable us to provide information concerning the characteristics of the haemophilia patient population in Austria and the actual treatment modalities used.

Calibrated automated thrombin generation in paediatric patients with inflammatory bowel disease.

UNLABELLED: In adults, inflammatory bowel disease (IBD) is associated with an increased risk of thromboembolic complications. The pathogenesis of IBD is not really clear and a high thrombin activity might contribute to disease progression. We wanted to see whether children with IBD have a higher thrombin generation (TG). PATIENTS, MATERIAL, METHODS: Plasma samples were collected of 20 patients with IBD and of 60 healthy controls (age range from 10 to 19). TG was measured by means of Calibrated automated thrombography (CAT). The disease activity was estimated, using the Pediatric Crohn's Disease Activity Index (PCDAI) for Crohn's disease and the Pediatric Ulcerative Colitis Disease Activity Index (PUCAI) for Ulcerative Colitis. In addition, we investigated F1+F2, TAT, TFPI and fibrinogen. RESULTS: There was a significant increase of endogenous thrombin potential (ETP), lag time and time to peak in patients with IBD, while peak showed no difference to healthy controls. ETP and F1+F2 in children with IBD also showed a significant correlation with PCDAI (PUCAI) and fibrinogen. CONCLUSION: IBD in children is associated with high TG, but this seems to be caused mainly by the inflammatory process and not by any individual disposition.

No differences in support of thrombin generation by neonatal or adult platelets.

UNLABELLED: Newborns have, despite low clotting factors and poor in vitro platelet function, a well functioning hemostasis. The reason for this is still not completely clear. The aim of our study was to investigate whether phospholipids in neonatal platelets differ from those in adult platelets in their total amount, in their exposure on the platelet surface, and their effect on thrombin generation (TG). METHODS: Clotting times of newborn and adult platelet-rich plasma were measured. Effect of newborn and adult platelets on TG was measured by means of CAT (calibrated automated thrombography). In addition, the effect of newborn and adult platelets with or without stimulation by ionophor on TG was measured in a purified prothrombinase complex. Phosphatidylserine-exposure (PS) of newborn and adult platelets was measured by flow cytometry of annexin V binding. The amount of phospholipids (PL) was determined by means of mass spectrometry. RESULTS: Clotting times of platelet-rich plasma (PRP) of newborns stimulated with ionophor showed a significant lower reduction of clotting time than in adult PRP. No differences in the support of TG between neonatal and adult platelets were found in neonatal or adult plasma by means of CAT. In the purified system TG was increased by adding ionophor stimulated platelets but no difference was evident between stimulated newborn and adult platelets. Flow cytometric analysis showed no difference in annexin V binding between adult and newborn platelets. The results of mass spectrometry showed a very similar amount and pattern of PL of adult and newborns platelets. CONCLUSION: Our results do not provide any evidence that a different PL content or expression of neonatal platelets may alter TG in neonates.

Screening for haemorrhagic disorders in paediatric patients by means of a questionnaire.

AIM: In children, screening for haemorrhagic disorders is further complicated by the fact that infants and young children with mild disease in many cases most likely will not have a significant history of easy bruising or bleeding making the efficacy of a questionnaire even more questionable. PATIENTS, METHODS: We compared the questionnaires of a group of 88 children in whom a haemorrhagic disorder was ruled out by rigorous laboratory investigation to a group of 38 children with mild von Willebrand disease (VWD). Questionnaires about child, mother and father were obtained prior to the laboratory diagnosis on the occasion of routine preoperative screening. RESULTS: 23/38 children with mild VWD showed at least one positive question in the questionnaire, while 21/88 without laboratory signs showed at least one positive question. There was a trend to more specific symptoms in older children. Three or more positive questions were found only in VWD patients, but only in a few of the control group. The question about menstrual bleeding in mothers did not differ significantly. Sensitivity of the questionnaire for a hemostatic disorder was 0.60, while specifity was 0.76. The negative predictive value was 0.82, but the positive predictive value was only 0.52. CONCLUSIONS: Our small study shows, that a questionnaire yields good results to exclude a haemostatic disorder, but is not a sensitive tool to identify such a disorder.

[Relevance of a single dose of 270 microg/kg recombinant factor VIIa for the treatment of patients with haemophilia and inhibitors - Recommendations from the GTH experts]. / Stellenwert der Einzelgabe von 270 microg/kg rekombinantem Faktor VIIa in der Behandlung von Hämophilie-Patienten mit Hemmkörpern - Empfehlungen der Experten.

Recombinant factor VIIa (rFVIIa; NovoSeven) is, besides other indications, authorised for the treatment of bleeding episodes in patients with hereditary haemophilia A or B and inhibitors. Based on the results of three clinical studies, marketing authorisation was granted for the single dose of 270 microg/kg body weight rFVIIa for the treatment of mild to moderate bleeding episodes in patients with haemophilia A or B with inhibitors in March 2007. Thereupon, an expert group analysed the relevance of this additional treatment option for clinical routine. Compared with the repeated application of 90 microg/kg body weight rFVIIa, quality of life may be improved if the single dose of 270 microg/kg body weight rFVIIa reduces the number of injections. The single dose has a benefit for those patients who require several rFVIIa applications or who do not respond adequately to low doses. Moreover, patients with poor venous access or patients who fear injections or reject them (especially children) may benefit from the single dose. The prescription of 270 microg/kg body weight rFVIIa as a single dose instead of multiple dosing of 90 microg/kg body weight is basically an individual and indication-related decision.

Thrombin generation in paediatric patients with congenital heart disease. Determination by calibrated automated thrombography.

UNLABELLED: Thrombin generation was studied in paediatric patients with congenital heart disease (CHD) undergoing cardiac surgery using the calibrated automated thrombography (CAT) in terms of the lag time until the onset of thrombin formation, time to thrombin peak maximum (TTP), endogenous thrombin potential (ETP), and thrombin peak height. The suitability to determine the coagulation status of these patients was investigated. PATIENTS, MATERIAL, METHODS: CAT data of 40 patients with CHD (age range from newborn to 18 years) were compared to data using standard coagulation parameters such as prothrombin (FII), antithrombin (AT), tissue factor pathway inhibitor (TFPI), prothrombin fragment 1.2 (F 1.2), thrombin-antithrombin (TAT), activated partial thromboplastin time (aPTT), and prothrombin time (PT). RESULTS: A significant positive correlation was seen between ETP and FII (p < 0.01; r = 0.369), as well as between peak height and FII (p < 0.01; r = 0.483). A significant negative correlation was seen between ETP and TFPI values (p < 0.05; r = -0.225) while no significant correlation was seen between peak height and TFPI. A significant negative correlation was seen between F 1.2 generation and ETP (p < 0.05; r = -0.254) and between F 1.2 generation and peak height (p < 0.05; r = -0.236). No correlation was seen between AT and ETP or peak. CONCLUSIONS: CAT is a good global test reflecting procoagulatory and inhibitory factors of the haemostatic system in paediatric patients with CHD.

Thrombin generation before and after multicomponent blood collection.

The development of apheresis technology has increased efficiency in donor blood use by collecting specific blood components in several combinations. The question of donor safety raised by the contact of donor blood with foreign, only in part biocompatible surfaces remains. The aim of this study was to estimate the effect of multicomponent blood collection on thrombin generation performing an overall function test of coagulation. DONORS, METHODS: 26 blood donors were included. Per apheresis two units of platelets and one unit of RBCs were collected by two cell separators (Amicus and Trima Accel). Each donor underwent the procedure on both apheresis systems. Samples were collected before, immediately after, and 48 hours after apheresis. Thrombin generation was measured by means of calibrated automated thrombography (CAT). RESULTS: CAT-data changed only slightly and no significant changes were seen before, immediately after, and 48 hours after apheresis. The parameters did not differ significantly between the two different apheresis devices. CONCLUSION: No change in parameters of continuous thrombin generation occurred, suggesting that apheresis did not lead to severe alterations in the haemostatic system.

Enhanced thrombin generation in plasma of severe thrombocytopenic patients due to rFVIIa.

UNLABELLED: RFVIIa-enhanced thrombin generation has been shown to be dependent on platelets. In previous work we have shown that addition of monocytes and rFVIIa to microparticle free plasma causes a distinct thrombin generation. The aim of our study has been to examine whether there is enough surface provided by microparticles in thrombocytopenic plasma to allow an effect of rFVIIa. PATIENTS, METHODS: Thrombin generation was measured in platelet rich plasma (PRP) and microparticle free plasma (MFP) of thrombocytopenic haemato-oncological patients with and without addition of rVIIa by means of calibrated automated thrombography. Microparticles were analyzed in PRP by FACS flow cytometry. RESULTS: Microparticle free plasma showed no thrombin generation with or without addition of rFVIIa. Addition of rFVIIa to PRP of thrombocytopenic patients led to a significant shortening of lag time and time to peak in thrombin generation, while ETP and peak remained unchanged. CONCLUSION: Our results show that even in plasma of severe thrombocytopenic patients enough surface may be provided by microparticles to allow an enhancement of thrombin generation by rFVIIa.