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Objectives To compare the diagnostic value of ultrashort echo time (UTE) magnetic resonance imaging (MRI) for the lung versus the gold standard computed tomography (CT) and two T1-weighted MRI sequences in children. Methods Twenty-three patients with proven oncologic disease (14 male, 9 female; mean age 9.0 + / − 5.4 years) received 35 low-dose CT and MRI examinations of the lung. The MRI protocol (1.5-T) included the following post-contrast sequences: two-dimensional (2D) incoherent gradient echo (GRE; acquisition with breath-hold), 3D volume interpolated GRE (breath-hold), and 3D high-resolution radial UTE sequences (performed during free-breathing). Images were evaluated by considering image quality as well as distinct diagnosis of pulmonary nodules and parenchymal areal opacities with consideration of sizes and characterisations. Results The UTE technique showed significantly higher overall image quality, better sharpness, and fewer artefacts than both other sequences. On CT, 110 pulmonary nodules with a mean diameter of 4.9 + / − 2.9 mm were detected. UTE imaging resulted in a significantly higher detection rate compared to both other sequences (p p Conclusion The UTE technique for lung MRI is favourable in children with generally high diagnostic performance compared to standard T1-weighted sequences as well as CT. Key Points • Due to the possible acquisition during free-breathing of the patients, the UTE MRI sequence for the lung is favourable in children. • The UTE technique reaches higher overall image quality, better sharpness, and lower artefacts, but not higher contrast compared to standard post-contrast T1-weighted sequences. • In comparison to the gold standard chest CT, the detection rate of small pulmonary nodules small nodules ≤ 4 mm and subtle parenchymal areal opacities is higher with the UTE imaging than standard T1-weighted sequences.

The terminal complement split product C5a has been described as an important mediator in inflammatory diseases. C5a is generated upon cleavage of C5 and earlier research suggests that, besides the known C5 convertases formed upon activation of the complement pathways, various enzymes could activate C5 directly. We demonstrate that eculizumab effectively blocks C5 activation when mediated by C5-convertase formation, but fails to block C5a generation resulting from direct enzymatic cleavage by trypsin and thrombin. C5a generated by these enzymes is shown to be fully biologically functional and can be blocked by IFX-1, a specific monoclonal anti-human C5a antibody. We further report clinical cases of atypical hemolytic uremic syndrome (aHUS) and C3 Glomerulonephritis (C3GN) patients under treatment with eculizumab presenting substantially elevated C5a levels. Thus, blocking the C5 convertase mediated activation of C5 may not be efficient to control C5a-mediated effects in human disease and that a targeted approach is warranted.

SummaryThe haemophilia treatment centre of the Clinic for Children and Youth Medicine in Jena extends medical care by health-promotion measures, namely: health counselling, adjuvant exercise therapy and school sports. In addition to the regular medical checks at the treatment centre patients are examined regarding physical fitness, joint situation, quality of life in general and disease-specific manner, as well as psycho-social and nutritional behaviour. Findings and medical results of the examinations are integrated into an individual advice on therapy, school sports, and health recommendations. This aimed at strengthening health-related resources and minimizing potential injuries. First long-term evaluation shows an increase of activity behaviour and physical fitness without increasing bleeding rate and maintained joint function. Conclusion: Combining functional prevention diagnostics and individual health counselling shows signs of improved patient’s health knowledge, self-competence and physical fitness.

In 2005 the Kompetenznetz Hämorrhagische Diathese Ost published epidemiologic data about patients with haemophilia A (HA) and haemophilia B (HB) in the eastern part of Germany. This study provides data about the development of treatment in these patients over the past 10 years. Data from 12 haemophilia centres in eastern Germany were retrospectively collected for the year 2015 from patients' records. We evaluated 413 patients (115 children, 298 adults) with HA or HB. A total of 286 patients (69.2%) had severe haemophilia (patients with severe haemophilia, PWSH). Compared with 2005, the proportion PWSH on prophylaxis increased from 90% to 98.8% in children and from 64% to 80.2% in adults. The use of plasma-derived factor concentrates decreased from70% to 55.3% in children and to 55.1% in adults. Mean annual factor consumption in PWSH without inhibitor was higher in 2015 compared with 2005 (children with HA: 151,489 vs. 98,894; adults with HA: 217,151 vs. 151,394; children with HB: 105,200 vs. 64,256; adults with HB: 159,185 vs. 85,295). Median annualized bleeding (annualized bleeding rate, ABR) and joint bleeding rates (annualized joint bleeding rate, AJBR) in 2015 were 2 and 0 in children and 3 and 0 in adults, respectively. In 2015 only one child (1.2%) but 101 (53.2%) adults with severe haemophilia were anti-hepatitis C virus (anti-HCV) positive. The rate of anti-HCV positive patients with active hepatitis C dropped from 63.8% to 12.9%. Within the last decade more patients with severe haemophilia were switched to a prophylactic regimen going along with a moderate increase in factor consumption achieving a low ABR and AJBR. Im Jahr 2005 hat das Kompetenznetz Hämorrhagische Diathese Ost epidemiologische Daten über Patienten im Osten Deutschlands mit Hämophilie A (HA) und B (HB) publiziert. Diese Studie liefert Daten über die Entwicklung der Hämophiliebehandlung bei diesen Patienten in den letzten 10 Jahren. Daten von 12 Hämophiliezentren im Osten Deutschlands aus dem Jahr 2015 wurden retrospektiv aus den Patientenakten ausgewertet. Wir untersuchten 413 Patienten (115 Kinder, 298 Erwachsene) mit HA oder HB. 286 Patienten (69,2%) hatten eine schwere Hämophilie (PMSH). Im Vergleich zu 2005 ist der Anteil der PMSH mit Prophylaxe bei den Kindern von 90% auf 98.8% und bei den Erwachsenen von 64% auf 80,2% angestiegen. Der Verbrauch von plasmatischen Faktorenkonzentraten ist von70% auf 55,3% bei Kindern und 55,1% bei Erwachsenen gesunken. Der mittlere Faktorenverbrauch bei PMSH ohne Hemmkörper war 2015 höher als 2005 (Kinder mit HA 151.489 versus 98.894, Erwachsene mit HA 217.151 versus 151.394, Kinder mit HB 105.200 versus 64.256, Erwachsenen mit HB 159.185 versus 85.295). Die mediane jährliche Blutungsrate und Gelenkblutungsrate in 2015 war 2 bzw. 0 bei Kindern und 3 bzw. 0 bei Erwachsenen. Im Jahr 2015 war lediglich ein Kind (1,2%) aber 101 (53,2%) Erwachsene mit schwerer Hämophilie anti-HCV positiv. Der Anteil der anti-HCV-positiven Erwachsenen mit aktiver Hepatitis C fiel von 63,8% auf 12,9%. In den letzten 10 Jahren wurden mehr Patienten auf eine prophylaktische Behandlung umgestellt. Dies geht mit einem moderaten Anstieg des Verbrauchs an Faktorenkonzentraten einher, resultiert aber in einer niedrigen Blutungsrate.

Multiple blood cell transfusions may cause iron overload or even liver fibrosis, requiring early diagnosis and intervention. SF is the standard for estimating iron levels in the body, but it also increases with inflammation. We hypothesized that T2 * magnetic resonance (MR) relaxometry is a more accurate alternative for follow-up in pediatric patients before and after allogenic SCT. Twenty-three children (mean age 10.2 years, 10 female, 13 male) were evaluated prospectively before SCT as well as at least 1 year after SCT with T2 * relaxometry on a 1.5 T MR-scanner to estimate liver iron concentrations from the T2 * values ("MR-Fe"). The results were compared with SF, while also considering CRP, and correlated with the number of transfusions. Overall, 24.3 transfusions were administered in average, mainly within 100 days of SCT (mean 10.5 units). Both MR-Fe and SF increased after SCT and decreased in the absence of new transfusions 1 year later without chelate therapy. This suggests regeneration of LP and iron loss, although the original states were not reached. Additionally, simultaneous peaks of CRP and SF were observed directly after SCT. MR-Fe did neither reveal these peaks nor was it associated with CRP (P = .39). We postulate that these early CRP and SF peaks after SCT are probably related to inflammatory reactions and not to iron overload. Thus, SF is not reliable for iron overload diagnosis after SCT in every condition. Beside this interaction, SF and MR-Fe revealed similar accuracy. MRI, however, has practical and economical disadvantages in routine estimation of iron.

Similar to the general population, overweight and obesity have increasingly become a medical and economic burden also in patients with haemophilia in industrialized nations. In this study in seven German haemophilia centres, we identified a prevalence of overweight and obesity of 25.2% among 254 young patients

Summary Various complement-mediated renal disorders are treated currently with the complement inhibitor eculizumab. By blocking the cleavage of C5, this monoclonal antibody prevents cell damage caused by complement-mediated inflammation. We included 23 patients with atypical haemolytic uraemic syndrome (aHUS, n = 12), C3 glomerulopathies (C3G, n = 9) and acute antibody-mediated renal graft rejection (AMR, n = 2), treated with eculizumab in 12 hospitals in Germany. We explored the course of complement activation biomarkers and the benefit of therapeutic drug monitoring of eculizumab. Complement activation was assessed by analysing the haemolytic complement function of the classical (CH50) and the alternative pathway (APH50), C3 and the activation products C3d, C5a and sC5b-9 prior to, 3 and 6 months after eculizumab treatment. Eculizumab concentrations were determined by a newly established specific enzyme-linked immunosorbent assay (ELISA). Serum eculizumab concentrations up to 1082 μg/ml point to drug accumulation, especially in paediatric patients. Loss of the therapeutic antibody via urine with concentrations up to 56 μg/ml correlated with proteinuria. In aHUS patients, effective complement inhibition was demonstrated by significant reductions of CH50, APH50, C3d and sC5b-9 levels, whereas C5a levels were only reduced significantly after 6 months' treatment. C3G patients presented increased C3d and consistently low C3 levels, reflecting ongoing complement activation and consumption at the C3 level, despite eculizumab treatment. A comprehensive complement analysis together with drug monitoring is required to distinguish mode of complement activation and efficacy of eculizumab treatment in distinct renal disorders. Accumulation of the anti-C5 antibody points to the need for a patient-orientated tailored therapy.

Background: Antithrombin [AT]-, protein C [PC]- or protein S [PS]-deficiency [D] constitutes a major risk factor for venous thromboembolism [VIE]. Primary study objective was to evaluate if the clinical presentation at first VTE onset differs between children and adults and to compare the individual recurrence risk among patients with respect to age at onset and their thrombophilia status ATD, PCD or PSD. Methods/Patients/Results: In 137 of 688 consecutively enrolled pediatric and adult VTE patients we calculated the absolute risk of VTE recurrence and event-free-survival adjusted for thrombophilia and positive family VTE history. At first VTE children manifested i) with a lower rate of pulmonary embolism, ii) a higher rate of cerebral vascular events or multiple VIES, and showed a higher proportion of unprovoked VIE compared to adolescents and adults. Adult patients reported more often a positive VIE history compared to younger study participants. The adjusted odds of recurrence in adults was 2.05 compared to children. Conclusion: At disease manifestation children and adults differ with respect to i) thrombotic locations, ii) percentage of unprovoked versus provoked VIE, and iii) different rates of positive VTE family histories. Furthermore, adults showed a two-fold increase risk of VTE recurrence compared to children. (C) 2016 ELSEVIER. All rights reserved.

The Epstein-Barr virus (EBV) produces different microRNAs (miRNA) with distinct regulatory functions within the infectious cycle. These viral miRNAs regulate the expression of viral and host genes and have been discussed as potential diagnostic markers or even therapeutic targets, provided that the expression profile can be unambiguously correlated to a specific stage of infection or a specific EBV-induced disorder. In this context, miRNA profiling becomes more important since the roles of these miRNAs in the pathogenesis of infections and malignancies are not fully understood. Studies of EBV miRNA expression profiles are sparse and have mainly focused on associated malignancies. This study is the first to examine the miRNA profiles of EBV reactivation and to use a correction step with seronegative patients as a reference. Between 2012 and 2017, we examined the expression profiles of 11 selected EBV miRNAs in 129 whole blood samples from primary infection, reactivation, healthy carriers and EBV seronegative patients. Three of the miRNAs could not be detected in any sample. Other miRNAs showed significantly higher expression levels and prevalence during primary infection than in other stages; miR-BHRF1-1 was the most abundant. The expression profiles from reactivation differed slightly but not significantly from those of healthy carriers, but a specific marker miRNA for each stage could not be identified within the selected EBV miRNA targets.

Children receiving multiple blood cell transfusions are prone to iron overload and successive tissue damage in liver parenchyma, making noninvasive screening options desirable. Ultrasound (US) elastography using acoustic radiation force impulse (ARFI) imaging enables evaluation of liver parenchyma stiffness, and MRI allows for quantification of liver iron concentration.The objective was to correlate US elastography with MRI in children who had undergone bone marrow transplantation and to evaluate the modification of liver tissue with US in combination with clinical parameters at follow-up.ARFI, T2*-weighted MRI and a clinical score (HepScore, based on parameters of liver function) were performed in 45 patients (24 male; mean age 9.7 years) before and 100 days and 365 days after transplantation. All received multiple blood transfusions (mean number 22.2 up until 1 year after transplantation). We correlated US findings and HepScore with MRI findings.We observed signs of iron accumulation in 29/45 (64.4%) patients on MRI (T2*10 ms) and 15/45 (33.3%) showed increased tissue stiffness (ARFI5.5 kPa). Correlation of elastography and MRI was not significant (P=0.57; n=51 matched measurements). Comparing US elastography with HepScore in receiver operating characteristic (ROC) curve analysis indicated a cut-off for affected parenchyma if HepScore was5 points (sensitivity 67%, specificity 68%). Simultaneous increases of both indicated tissue alteration.Combining US and HepScore enabled detection of liver tissue alteration through iron overload, but we found no direct significant effect of estimated iron from MRI on ARFI imaging.

Recently, variants in DONSON have been reported to cause different disorders of the microcephalic primordial dwarfism spectrum. Using whole-exome sequencing, we identified two novel, compound heterozygous DONSON variants in a pair of siblings, one of whom was previously diagnosed with Fanconi anemia. This occurred because the present cases exhibited clinical findings in addition to those of the microcephalic primordial dwarfism disorder, including severe limb malformations. These findings suggest that the DONSON and Fanconi anemia proteins could have supplementary roles in developmental processes as they have in the maintenance of genomic integrity, resulting in related disease phenotypes.

The haemophilia treatment centre of the Clinic for Children and Youth Medicine in Jena extends medical care by health-promotion measures, namely: health counselling, adjuvant exercise therapy and school sports. In addition to the regular medical checks at the treatment centre patients are examined regarding physical fitness, joint situation, quality of life in general and disease-specific manner, as well as psycho-social and nutritional behaviour. Findings and medical results of the examinations are integrated into an individual advice on therapy, school sports, and health recommendations. This aimed at strengthening health-related resources and minimizing potential injuries. First long-term evaluation shows an increase of activity behaviour and physical fitness without increasing bleeding rate and maintained joint function.Combining functional prevention diagnostics and individual health counselling shows signs of improved patient's health knowledge, self-competence and physical fitness.Das Behandlungskonzept am Hämophilie-Behandlungszentrum der Kinder- und Jugendklinik in Jena wurde um gesundheitsförderliche Maßnahmen der Gesundheitsberatung, adjuvanten Bewegungstherapie und des Schulsports erweitert. Begleitend zur turnusmäßigen Untersuchung werden Patienten zusätzlich hinsichtlich körperlicher Leistungs - fähigkeit, aktueller Gelenksituation, allgemeiner und krankheitsspezifischer Lebensqualität sowie psychosozialen Verhaltens untersucht. Ferner wird das Ernährungsverhalten der Patienten erfasst und eine befundbasierte Gesundheitsberatung angeboten. Die Ergebnisse fließen zusammen mit den klinischen Befunden in individuelle Therapie-, Schulsport- und Gesundheitsempfehlungen ein. Eine erste Längsschnittauswertung weist auf eine Zunahme des Aktivitätsverhaltens und der körperlichen Leistungsfähigkeit bei gleichzeitig stabiler Blutungsrate und Erhaltung der Gelenkfunktion hin.Die Kombination von funktioneller Präventionsdiagnostik und individueller Gesundheitsberatung mit besonderer Förderung des Gesundheitswissens, der Selbstkompetenz und der körperlichen Aktivität haben sich bislang bewährt.

Safety, tolerability, and pharmacokinetics of recombinant ADAMTS-13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13; BAX 930; SHP655) were investigated in 15 patients diagnosed with severe congenital ADAMTS-13 deficiency (plasma ADAMTS-13 activity

Acquired thrombotic thrombocytopenic purpura (TTP) is the consequence of a severe ADAMTS13 deficiency resulting from autoantibodies inhibiting ADAMTS13 or accelerating its clearance. Despite the success of plasma exchange the risk of relapse is high. From 2 patients (A and B), splenectomized for recurrent episodes of acquired TTP, the splenic B-cell response against ADAMTS13 was characterized through generation of human monoclonal anti-ADAMTS13 autoantibodies (mAbs) by cloning an immunoglobulin G (IgG)4κ- and IgG4λ-Fab library using phage display technology and by Epstein-Barr virus transformation of switched memory B cells (CD19+/CD27+/IgG+). Sequence analysis of the anti-ADAMTS13 IgGs of both patients revealed that the VH gene use was limited in our patients to VH1-3 (55%), VH1-69 (17%), VH3-30 (7%), and VH4-28 (21%) and contained 8 unique and thus far not reported heavy-chain complementarity determining region 3 motifs, of which 4 were shared by the 2 patients. The discovery of several highly similar anti-ADAMTS13 autoantibodies in 2 unrelated TTP patients suggests that the autoimmune response is antigen driven, because the probability that such similar immunoglobulin rearrangements happen by chance is very low (< 10(-9)).

SummaryThe Upshaw Schulman syndrome (MIM #274150) is a hereditary deficiency of the von Willebrand factor cleaving protease (ADAMTS13) due to homozygous or compound heterozygous mutations in the ADAMTS13 gene. Patients are prone to bouts of thrombotic thrombocytopenic purpura. However, disease manifestation needs a second trigger event. Pregnancy is a known risk factor for TTP. Patients with USS may manifest during pregnancy and the postpartum period or relapse with a TTP bout. Before plasma therapy mortality for both the mother and the fetus was high, but even nowadays when plasma is delivered, therapy is challenging, still bearing a high risk for miscarriage or long term sequelae for the mother.In this report on pregnancies in three mothers with USS, plasma therapy was increased in frequency and amount given with regard to platelet count or ADAMTS13 activity, thus leading to a successful outcome.

Objectives Thrombocytopenia occurs in pediatric patients after SCT and has to be treated with platelet transfusions which bear certain risks and represent a significant cost factor. Monitoring immature platelet (IPF) fraction has been proposed to predict platelet recovery thereby reducing the need for transfusions. Design and methods Hematological parameters including IPF were systematically studied in 17 pediatric patients after either peripheral blood or bone marrow stem cell transplantation. Results Time to platelet recovery depended on the source of stem cells while no differences were detected between percentaged IPF peak concentration and time between IPF peak concentration and platelet recovery between the groups. Correlation between the timepoints of percentaged IPF peak and platelet recovery was high but large interindividual differences were observed concerning the duration of this period. In addition, in some patients high IPF concentrations were not followed by platelet recovery. Conclusions Although in general high IPF concentrations are followed by platelet recovery wide interindividual variations exist and even no recovery was recorded in four patients. As the latter children are not readily identifiable beforehand IPF should not be used to omit platelet transfusions.

SummaryIntroduction: Desmopressin (DDAVP) testing (DT) in patients (pts) with haemophilia A (HA) and carriers (CHA) is up to now not standardized. This prompted us to evaluate results of DT carried out between 1996 and 2011 in centres of the Competence Network Haemor-rhagic Diatheses East. Patients and method: An increase of the factor VIII activity (FVIII) above 50% or at least the two fold of initial values within 120 min after DDAVP was defined as complete response (CR). Data from 80 patients (31 children, 49 adults) of whom 64 suffered from HA (sub-HA: n = 48; mild: n = 14; moderate: n = 2) and 16 patients CHA were evaluated. Results: In 34 patients DDAVP was given i. v. (dose range: 0.26–0.6 μg/kg body weight, mean: 0.33), in 31 intranasally (i.n. 300–600 μg) and in 15 s. c. (15–40 μg). The maximal FVIII increase was reached 60 min after DDAVP. For i. v. application the mean FVIII increase was 3.1-fold, for i. n. 2.1-fold and for s. c. 2.4-fold. A CR was de tected in 71 patients, a non-response in 9. Mild side effects such as flush, headaches or nausea were observed in 11 patients (14%). Conclusion: For desmopressin testing in patients with haemophilia A and carriers i. v. application at 0.3 μg/kg body weight and the determination of FVIII before and 60 min after desmopressin infusion is recommended.

Thrombotic microangiopathies (TMA) represent a spectrum of related disorders associated with newly formed thrombi that block perfusion and thus affect the function of either renal or neurological organs and tissue. Recent years have seen a dramatic development in the field of TMA and for the two major forms hemolytic uremic syndrome (HUS) and thrombocytopenic purpura (TTP), new genetic causes and also autoimmune forms have been identified. This development indicates a similar pathophysiology and suggests that the two acute disorders are based on common principles. HUS is primarily a kidney disease and TTP also develops in the kidney and at neurological sites. In HUS thrombi formation is likely due to a deregulated complement activation and inappropriate platelet activity. In TTP thrombi formation occurs because of inappropriate processing of released multimers of von Willebrand Factor (vWF). Defining both the similarities and the unique features of each disorder will open up new ways and concepts that are relevant for diagnosis, for therapy, and for the prognostic outcome of kidney transplantations. Here we summarize the most relevant topics and timely issues that were presented and discussed at the 4th International Workshop on Thrombotic Microangiopathies held in Weimar in October 2009 (www.hus-ttp.de).

Fungal infections are a leading cause of morbidity and mortality in severely immunocompromised patients and have been increasing in incidence in recent years. Invasive aspergillosis (IA) is the most common filamentous fungal infection and is, in adults as well as in children, difficult to diagnose. Several PCR assays to detect Aspergillus DNA have been established, but so far, studies on molecular tools for the diagnosis of IA in children are few. We evaluated the results of a nested PCR assay to detect Aspergillus DNA in clinical samples from paediatric and adolescent patients with suspected IA. Blood and non-blood samples from immunocompromised paediatric and adolescent patients with suspected invasive fungal infection were sent for processing Aspergillus PCR to our laboratory. PCR results from consecutive patients from three university children's hospitals investigated between November 2000 and January 2007 were evaluated. Fungal infections were classified according to the EORTC classification on the grounds of clinical findings, microbiology and radio-imaging results. Two hundred and ninety-one samples from 71 patients were investigated for the presence of Aspergillus DNA by our previously described nested PCR assay. Two, 3 and 34 patients had proven, probable and possible IA, respectively. Sensitivity (calculated from proven and probable patients, n=5) and specificity (calculated from patients without IA, n=32) rates of the PCR assay were 80 and 81 %, respectively. Our nested PCR assay was able to detect Aspergillus DNA in blood, cerebrospinal fluid and bronchoalveolar lavage samples from paediatric and adolescent patients with IA with high sensitivity and specificity rates.

SummaryVon Willebrand factor (VWF) and related parameters as well as the protease activity regulating its biological activity were measured in plasma of healthy controls and patients with different cause and severity of systemic inflammation to examine the efficacy of the measures to detect highly prothrombotic states including thrombotic microangiopathy (TMA), one of the sequelae of sepsis. Plasma levels of VWF increased with increasing severity of systemic inflammation, probably due to activation of the endothelium. In parallel, the proteolytic activity of VWF inactivating protease, ADAMTS13, stepwise declined with the severity of inflammation, emphasizing the role of VWF-triggered platelet aggregation on the endothelium subsequently followed by development of TMA. As a consequence, the ratio of VWF antigen level and ADAMTS13 activity was significantly higher in patients with inflammation and sepsis, suggesting that this ratio might be more useful for the diagnosis of highly prothrombotic states including TMA than VWF multimer analysis alone. These findings suggest that ADAMTS13, VWF and related parameters, even in a combined approach, might be useful for the diagnosis and the therapeutic monitoring of patients with sepsis associated thrombotic microangiopathy.

Vascular complications remain the most common cause of early renal allograft loss in patients with end-stage renal failure. Underlying thrombophilic disorders increase the risk of early graft thrombosis. A male adolescent with high-risk thrombophilia because of combined heterozygous factor V Leiden (G1691A) and prothrombin gene (G20210A) mutation developed HIT II. Hemodialysis and subsequent renal transplantation were undertaken using recombinant hirudin, a direct and selective thrombin inhibitor, as an anticoagulant. Primary function in the transplanted kidney was excellent. No thrombotic or hemorrhagic events have occurred and follow-up showed excellent long-term graft survival. Patients on HD have an increased risk for the development of HIT, and therefore, they need repetitive screening for the development of acquired thrombotic risk factors (e.g. HIT II or lupus anticoagulant). R-hirudin is efficacious and safe on both HD and following renal transplantation.

Various diseases shift the composition of human plasma; hence, the relative quantification of plasma constituents offers the opportunity to use the dynamic and complex composition of plasma to gain information on novel diagnostic and prognostic factors. Since plasma contains, besides water, mostly proteins, UV-resonance Raman spectroscopy (UVRR) seems to be a suitable method for investigating plasma. With this method the signals of aromatic amino acids and proteins are selectively enhanced. In this study an UV-resonance Raman approach was used for the investigation of human plasma of healthy volunteers and patients with thrombotic microangiopathy. For comparison, selected plasma components were analyzed for a more detailed characterization of cryoprecipitates from human plasma.

Precise assessment of lymph nodes is crucial to the choice of therapy and prediction of outcome in cases of malignancy. Electrical impedance scanning (EIS) is being experimentally investigated for potential use as a diagnostic tool for differentiation of malignant lesions. Malignancies show different electrical properties with changes in conductivity and capacitance that can be analysed by EIS. Using a TransScan TS-2000 (TransScan Medical, Migdal Ha'Emek, Israel, distributed by Siemens-Elema AB, Solna, Sweden), EIS has been used in various studies for the identification of breast cancer as well as for characterisation of superficial lesions. To evaluate the reliability of EIS for classifying lymph nodes in a pediatric population with sonographically suspicious lesions and to prove its accuracy. The study population consisted of 77 children (42 boys, 35 girls) aged 1.1–17.1 years. All EIS results were compared to either histopathological findings or long-term follow-up investigations. Sensitivity for malignancies using EIS was 75% and specificity was 87%. The negative predictive value was 93% and the positive predictive value was 60%. This study suggests the potential usefulness of EIS as an additional imaging modality for the differentiation of lymph-node diseases in children. The histopatholgical spectrum of malignant lymph node transformation in children compared to studies in adults, and the characteristic meltdown in inflammatory or granulomatous transformed nodes, pose challenges to differentiation based on sonographic evaluation, and also to EIS classification.

Background The present investigation was intended to further clarify the mechanisms involved in renal dysfunction following high-dose methotrexate (HD-MTX) treatment. Patients and Methods Fifty eight predominately pediatric patients [39 male, 19 female; mean age 12.3 years (range 2.2–34.1)] suffering from acute lymphoblastic leukemia (ALL, n = 28), Non Hodgkins lymphoma (NHL, n = 13), osteosarcoma (n = 8), malignant brain tumor (n = 6), or an ALL relapse (n = 3), were prospectively examined. In the course of 220 infusions of HD-MTX, glomerular and tubular renal function was determined by measuring proteinuria and glomerular filtration rate (GFR), as well as renal excretion of alpha-1-microglobulin (AMG) and N-acetyl-β-D-glucosaminidase (NAG). It was investigated whether there were differences in MTX toxicity in dependence on the administered dose (1, 5, or 12 g/m2 BSA), on the combination with other cytostatic agents (ifosfamide or cyclophosphamide), on the metabolism of MTX into 7-OH-MTX, and on pre-treatment with MTX. Results The administration of HD-MTX has no direct tubulotoxic effect. The disturbance in glomerular function was dose dependently and indicated by an increase in proteinuria as well as by a decrease in GFR; all changes were completely reversible and did not correlate to the metabolism of MTX to 7-OH-MTX. Increasing the number of MTX therapeutic cycles did not increase the nephrotoxicity of MTX. Conclusion MTX is not directly tubulotoxic. Its side effects on glomeruli are usually without clinical relevance. Med Pediatr Oncol 2003;40:348–354. © 2003 Wiley-Liss, Inc.

Haemophagocytic lymphohistiocytosis (HLH) in the context of malignancy is mainly considered a challenge of adult haematology. While this association is also observed in children, little is known regarding inciting factors, appropriate treatment and prognosis. We retrospectively analysed 29 paediatric and adolescent patients for presenting features, type of neoplasm or preceding chemotherapy, treatment and outcome. Haemophagocytic lymphohistiocytosis was considered triggered by the malignancy (M-HLH) in 21 patients, most of whom had T- (n = 12) or B-cell neoplasms (n = 7), with Epstein-Barr virus as a co-trigger in five patients. In eight patients, HLH occurred during chemotherapy (Ch-HLH) for malignancy, mainly acute leukaemias (n = 7); an infectious trigger was found in seven. In M- and Ch-HLH, median overall survival was 1·2 and 0·9 years, and the 6 month survival rates were 67% and 63%, respectively. Seven of 11 deceased M-HLH patients exhibited active malignancy and HLH at the time of death, while only two out of five deceased Ch-HLH patients had evidence of active HLH. To overcome HLH, malignancy- and HLH-directed treatments were administered in the M-HLH cohort; however, it was not possible to determine superiority of one approach over the other. For Ch-HLH, treatment ranged from postponement of chemotherapy to the use of etoposide-containing regimens.

Acquired thrombotic-thrombocytopenic purpura (TTP) is an autoimmune disorder characterized by autoantibodies directed against the von Willebrand metalloprotease. Depletion of B-cells can prevent synthesis of this antibody and presumably induce remission of the disease. In adults, Rituximab (RTX) was effective in relapsed or refractory acute idiopathic TTP.We report the long-term follow-up of five children and two adolescents (age at diagnosis 6-19 years, median 15 years) who were treated with RTX for recurrent or refractory TTP. Some of the patients suffered from recurrent refractory TTP with long histories of previous unsuccessful treatments. One had TTP associated with pancreatitis.Three patients have been in complete remission after one treatment course with RTX. Four relapsed after 1 to 5 years, respectively, and responded to additional courses of RTX. One of them is in long-term remission after a third course of RTX and splenectomy. Compared to literature reports with a median follow up of 1.4 years (3-46 month), follow-up of our patients after treatment with RTX was very long (2-12.7 years, median 7.7 years). RTX therapy could induce long-term remissions in children with refractory recurrent TTP. Median duration of remission was longer and relapses per patient-years less frequent in patients receiving RTX compared to patients not receiving it. Remissions were achieved in children within one week, much faster than in adults.Because of the rapid induction of remissions, RTX may be suitable for first-line therapy in pediatric acquired antibody-mediated TTP.

Wiskott-Aldrich syndrome (WAS) is caused by mutations in the WAS gene and is characterized by immunodeficiency, eczema and microthrombocytopenia. The molecular link between WAS mutations and microthrombocytopenia is unknown. Profilin1 (Pfn1) is a key actin-regulating protein that, besides actin, interacts with phosphoinositides and multiple proline-rich proteins, including the WAS protein (WASp)/WASp-interacting protein (WIP) complex. Here we report that mice with a megakaryocyte/platelet-specific Pfn1 deficiency display microthrombocytopenia due to accelerated turnover of platelets and premature platelet release into the bone marrow. Both Pfn1-null mouse platelets and platelets isolated from WAS patients contained abnormally organized and hyperstable microtubules. These results reveal an unexpected function of Pfn1 as a regulator of microtubule organization and point to a previously unrecognized mechanism underlying the platelet formation defect in WAS patients.

In chronic granulomatous disease allogeneic haemopoietic stem-cell transplantation (HSCT) in adolescents and young adults and patients with high-risk disease is complicated by graft-failure, graft-versus-host disease (GVHD), and transplant-related mortality. We examined the effect of a reduced-intensity conditioning regimen designed to enhance myeloid engraftment and reduce organ toxicity in these patients.This prospective study was done at 16 centres in ten countries worldwide. Patients aged 0-40 years with chronic granulomatous disease were assessed and enrolled at the discretion of individual centres. Reduced-intensity conditioning consisted of high-dose fludarabine (30 mg/m(2) [infants9 kg 1·2 mg/kg]; one dose per day on days -8 to -3), serotherapy (anti-thymocyte globulin [10 mg/kg, one dose per day on days -4 to -1; or thymoglobuline 2·5 mg/kg, one dose per day on days -5 to -3]; or low-dose alemtuzumab [1 mg/kg on days -8 to -6]), and low-dose (50-72% of myeloablative dose) or targeted busulfan administration (recommended cumulative area under the curve: 45-65 mg/L × h). Busulfan was administered mainly intravenously and exceptionally orally from days -5 to -3. Intravenous busulfan was dosed according to weight-based recommendations and was administered in most centres (ten) twice daily over 4 h. Unmanipulated bone marrow or peripheral blood stem cells from HLA-matched related-donors or HLA-9/10 or HLA-10/10 matched unrelated-donors were infused. The primary endpoints were overall survival and event-free survival (EFS), probabilities of overall survival and EFS at 2 years, incidence of acute and chronic GVHD, achievement of at least 90% myeloid donor chimerism, and incidence of graft failure after at least 6 months of follow-up.56 patients (median age 12·7 years; IQR 6·8-17·3) with chronic granulomatous disease were enrolled from June 15, 2003, to Dec 15, 2012. 42 patients (75%) had high-risk features (ie, intractable infections and autoinflammation), 25 (45%) were adolescents and young adults (age 14-39 years). 21 HLA-matched related-donor and 35 HLA-matched unrelated-donor transplants were done. Median time to engraftment was 19 days (IQR 16-22) for neutrophils and 21 days (IQR 16-25) for platelets. At median follow-up of 21 months (IQR 13-35) overall survival was 93% (52 of 56) and EFS was 89% (50 of 56). The 2-year probability of overall survival was 96% (95% CI 86·46-99·09) and of EFS was 91% (79·78-96·17). Graft-failure occurred in 5% (three of 56) of patients. The cumulative incidence of acute GVHD of grade III-IV was 4% (two of 56) and of chronic graft-versus-host disease was 7% (four of 56). Stable (≥90%) myeloid donor chimerism was documented in 52 (93%) surviving patients.This reduced-intensity conditioning regimen is safe and efficacious in high-risk patients with chronic granulomatous disease.None.

Clinical and genetic heterogeneity renders confirmation or exclusion of autoimmune lymphoproliferative syndrome difficult. To re-evaluate and improve the currently suggested diagnostic approach to patients with suspected FAS mutation, the most frequent cause of autoimmune lymphoproliferative syndrome, we prospectively determined 11 biomarkers in 163 patients with splenomegaly or lymphadenopathy and presumed or proven autoimmune cytopenia(s). Among 98 patients sequenced for FAS mutations in CD3(+)TCRα/β(+)CD4(-)CD8(-) "double negative" T cells, 32 had germline and six had somatic FAS mutations. The best a priori predictor of FAS mutations was the combination of vitamin B12 and soluble FAS ligand (cut-offs 1255 pg/mL and 559 pg/mL, respectively), which had a positive predictive value of 92% and a negative predictive value of 97%. We used these data to develop a web-based probability calculator for FAS mutations using the three most discriminatory biomarkers (vitamin B12, soluble FAS ligand, interleukin-10) of the 11 tested. Since more than 60% of patients with lymphoproliferation and autoimmune cytopenia(s) in our cohort did not harbor FAS mutations, 15% had somatic FAS mutations, and the predictive value of double-negative T-cell values was rather low (positive and negative predictive values of 61% and 77%, respectively), we argue that the previously suggested diagnostic algorithm based on determination of double-negative T cells and germline FAS sequencing, followed by biomarker analysis, is not efficient. We propose vitamin B12 and soluble FAS ligand assessment as the initial diagnostic step with subsequent decision on FAS sequencing supported by a probability-calculating tool. peerReviewed

The heterogeneity of primary and secondary immunodeficiencies demands for the development of a comprehensive flow cytometric screening system, based on reference values that support a standardized immunophenotypic characterization of most lymphocyte subpopulations.Peripheral blood samples from healthy adult volunteers (n = 25) were collected and split into eight panel fractions (100 µl each). Subsequently, premixed eight-color antibody cocktails were incubated per specific panel of whole blood to detect and differentiate cell subsets of: (i) a general lymphocyte overviews, (ii) B-cell subpopulations, (iii) CD4+ subpopulations, (iv) CD8+ subpopulations, (v) regulatory T-cells, (vi) recent thymic emigrants (RTE), (vii) NK-cell subpopulations, and (viii) NK-cell activation markers. All samples were lysed, washed, and measured by flow cytometry. FACS DIVA software was used for data analysis and calculation of quadrant statistics (mean values, standard error of mean, and percentile ranges).Whole blood staining of lymphocytes provided the analysis of: (i) CD3+, 4+, 8+, 19+, 16/56+, and activated CD4/8 cells; (ii) immature, naïve, nonswitched/switched, memory, (activated) CD21(low) , transitional B-cells, plasmablasts/plasmacells; (iii and iv) naïve, central memory, effector, effector memory, TH1/TH2/TH17-like, and CCR5+CD8-cells; (v) CD25+, regulatory T-cells (naïve/memory, HLA-DR+); (vi) α/β- and γ/δ-T-cells, RTE in CD4/CD8 cells; (vii) immature/mature CD56(bright) , CD94/NKG2D+ NK-cells; and (viii) Nkp30, 44, 46, and CD57+NK-cells. Clinical examples and quadrant statistics are provided.The present study represents a practical approach to standardize the immunophenotyping of most T-, B-, and NK-cell subpopulations. That allows differentiating whether abnormalities or developmental shifts observed in lymphocyte subpopulations originates either from primary or secondary immunological disturbance.

Routine assessment of body iron load in patients with acute leukemia is usually done by serum ferritin (SF) assay; however, its sensitivity is impaired by different conditions including inflammation and malignancy.To estimate, using MRI, the extent of liver iron overload in children with acute leukemia and receiving blood transfusions, and to examine the association between the degree of hepatic iron overload and clinical parameters including SF and the transfusion iron load (TIL).A total of 25 MRI measurements of the liver were performed in 15 children with acute leukemia (mean age 9.75 years) using gradient-echo sequences. Signal intensity ratios between the liver and the vertebral muscle (L/M ratio) were calculated and compared with SF-levels. TIL was estimated from the cumulative blood volume received, assuming an amount of 200 mg iron per transfused red blood cell unit.Statistical analysis revealed good correlation between the L/M SI ratio and TIL (r = -0.67, P = 0.002, 95% confidence interval CI = -0.83 to -0.34) in patients with acute leukemia as well as between L/M SI ratio and SF (r = -0.76, P = 0.0003, 95% CI = -0.89 to -0.52).SF may reliably reflect liver iron stores as a routine marker in patients suffering from acute leukemia.

SummaryThrombocytopenia during pregnancy is a common diagnostic and management problem. Several differential diagnosis must be considered including manifestations of thrombotic thrombocytopenic purpura (TTP). We report here on a case of a 21-year-old pregnant woman who presented initially severe thrombocytopenia (8 Gpt/l) in the 20th+1 week of gestation. The patient had an antibody against ADAMTS13, and enzyme activity was

There are no widely accepted criteria for the definition of hematopoietic stem cell transplant -associated microangiopathy (TAM). An International Working Group was formed to develop a consensus formulation of criteria for diagnosing clinically significant TAM.The participants proposed a list of candidate criteria, selected those considered necessary, and ranked those considered optional to identify a core set of criteria. Three obligatory criteria and four optional criteria that ranked highest formed a core set. In an appropriateness panel process, the participants scored the diagnosis of 16 patient profiles as appropriate or not appropriate for TAM. Using the experts' ratings on the patient profiles as a gold standard, the sensitivity and specificity of 24 candidate definitions of the disorder developed from the core set of criteria were evaluated. A nominal group technique was used to facilitate consensus formation. The definition of TAM with the highest score formed the finalThe Working Group proposes that the diagnosis of TAM requires fulfilment of all of the following criteria: (i)4% schistocytes in blood; (ii) de novo, prolonged or progressive thrombocytopenia (platelet count50 x 109/L or 50% or greater reduction from previous counts); (iii) sudden and persistent increase in lactate dehydrogenase concentration; (iv) decrease in hemoglobin concentration or increased transfusion requirement; and (v) decrease in serum haptoglobin. The sensitivity and specificity of this definition exceed 80%.The Working Group recommends that the presented criteria of TAM be adopted in clinical use, especially in scientific trials.

Invasive aspergillosis (IA), a complication with high mortality rates, especially in disseminated IA with cerebral involvement, is difficult to diagnose. Biopsy of cerebral lesions is often not feasible, and culture of Aspergillus spp. from cerebrospinal fluid (CSF) is frequently negative. New molecular methods have emerged for diagnosing IA. So far, there are only few reports of Aspergillus DNA detection in CSF. After modifying the DNA extraction protocol, we detected Aspergillus DNA in CSF samples by a previously described nested PCR assay. In six patients with hematologic malignancy and cerebral aspergillosis, CSF samples were investigated for Aspergillus DNA. IA was classified according to the EORTC/MSG 2002 criteria. Two patients each had proven, probable, and possible IA. Thirty-five CSF samples were investigated for Aspergillus DNA by nested PCR. Samples with positive results in the nested PCR assay were quantified by LightCycler PCR assay. Fourteen CSF samples showed positive results in the nested PCR assay. Of these, six samples gave positive results in real-time PCR. The range of CFU per ml was 2,154 to 63,100,000. The highest number of CFU per ml was found in a CSF sample of a patient with acute lymphocytic leukemia and probable cerebral aspergillosis. Detection of Aspergillus DNA in CSF samples is thus possible and has the potential to improve diagnosis of cerebral aspergillosis. Further prospective studies with larger numbers of patients must be performed to evaluate the clinical significance of Aspergillus PCR with CSF samples.

Transplantation-associated microangiopathy (TAM) is a severe complication of stem cell transplantation. Although TAM shares many features with idiopathic thrombotic thrombocytopenic purpura or hemolytic uremic syndrome, the prognosis of TAM is worse. Clinical similarities and the observation that uncleaved ultralarge von Willebrand factor (ULVWF) multimers are found in the circulation of patients suffering from TAM suggest a defect in VWF proteolysis that may be due to a deficiency in ADAMTS13 activity. In this study the course of 28 consecutive patients, who received an allogeneic stem cell transplant was correlated to ADAMTS13 activity. Before stem cell transplantation, mean ADAMTS13 activity was within normal range. Within the first 8 weeks, mean activity declined to less than half the baseline activity. Furthermore, most of the patients showed normalization of ADAMTS13 activity. Low levels of ADAMTS13 activity were not correlated with clinical signs of thrombotic microangiopathy. However, two patients with clinical TAM had the lowest activity of all patients when suffering a severe bout of microangiopathy. Plasma exchange was not able to normalize ADAMTS13 deficiency in these patients, suggesting inactivation or consumption of ADAMTS13 activity in TAM.

The study was performed to compare whole-body short time inversion recovery (STIR) MR imaging and (99m)Tc-methylene diphosphonate planar scintigraphy in the examination of children with suspected multifocal skeletal malignant lesions. Sixteen patients with known or suspected malignant skeletal disease underwent both whole-body STIR MR imaging and bone scintigraphy. The lesions were described and numbered according to scintigraphic evaluation criteria. Thus, 16 regions were analyzed in each patient for the comparison between the two modalities. Histology was proven in the primary malignant regions. Follow-up MRIs were registered. Scintigraphy and MRI follow-up were evaluated as gold standard. A total of 139 different lesions was observed by both modalities. Baseline whole-body MRI revealed 119 bone lesions in 256 possible sites (46.5%); scintigraphy revealed only 58 lesions (22.6%). Congruence was observed in only four patients (25%). According to the location of the lesion, correlation was observed in 39/139 lesions (28%). In all, 57.5% of the lesions were detected only by MRI and 14.5% of the lesions were detected only by scintigraphy. Whole-body MRI was more sensitive (P0.001). Of all lesions numbered which could be separated in the initial MRI, whole-body MRI detected 178 lesions in the patients. The results suggest that whole-body MRI using a STIR sequence is an effective radiation free method for examination of children with suspected multifocal bone lesions. MRI showed more lesions than conventional (99m)Tc-methylene diphosphonate scintigraphy. Therefore, whole-body MRI may be feasible as a screening modality for metastatic and skip lesions in osteosarcoma, PNET, Ewing sarcoma and Langerhans cell histiocytosis in children.

Priapism is defined as prolonged and persistent erection of the penis without sexual stimulation. It is associated with excessive hyperleukocytosis (e.g. in acute or chronic leukaemia); however, this complication is rarely seen in the pediatric population. We report a 12-year-old boy suffering from acute leukaemia presenting with, at first intermittent, but increasingly persistent erection. Doppler US revealed signs of high-flow priapism. MRI excluded intrapelvic tumour masses, and three-dimensional contrast-enhanced MR angiography could not demonstrate an arteriovenous fistula or thrombosis. Cavernosal blood-gas measurement was in agreement with high-flow priapism. On the basis of the imaging findings, invasive therapeutic management was avoided in our patient with a successful outcome.

Thrombotic thrombocytopenic purpura (TTP) is caused by the persistence of the highly reactive high-molecular-weight multimers of von Willebrand factor (VWF) due to deficiency of the specific VWF-cleaving protease (VWF-CP) ADAMTS13, resulting in microangiopathic disease. The acquired form is caused by autoantibodies against VWF-CP, whereas homozygous or compound heterozygous mutations of ADAMTS13 are responsible for recessively inherited TTP. We investigated 83 children with hemolytic or thrombocytopenic episodes with or without additional neurologic symptoms or renal failure. The presumed diagnosis was chronic idiopathic thrombocytopenic purpura (ITP; n = 50), TTP (n = 8), hemolytic uremic syndrome (HUS; n = 24), and Evans syndrome (n = 1). A severe deficiency of VWF-CP (≤ 5%) was found in all investigated patients with TTP and in none of those with HUS. Additionally, 2 of 50 patients with a prior diagnosis of ITP were deficient for VWF-CP. Antibodies against VWF-CP were found in 4 children. Mutation analysis of the ADAMTS13 gene in the patients deficient in VWF-CP by direct sequencing of all 29 exons identified 8 different mutations, suggesting the hereditary form of TTP in 1 patient with ITP, in the patient with Evans syndrome, and in 5 of the 8 patients with TTP. The phenotype of TTP in childhood can be rather variable. Besides the classical clinical picture, oligosymptomatic forms may occur that can delay the identification of patients at risk.

Abdominal problems often complicate the clinical course after bone marrow transplantation. Graft-versus-host disease occurs as a complication of allogenic bone marrow transplantation. In this report, the findings of intestinal involvement are described and correlated with histopathological findings. Increased bowel-wall thickness and increased vascularity were shown by US. MRI demonstrated generalised increased bowel-wall thickness associated with bowel-wall enhancement after administration of i.v. gadolinium.

The aim of this prospective study was to assess glomerular and tubular renal function before, and 1 and 2 years after hematological stem cell therapy (HSCT) in children and adolescents. 137 consecutive patients undergoing HSCT, for malignant diseases, were included in a prospective trial. Forty-four patients were followed for up to 1 year after HSCT and 36 for up to 2 years, without relapse. Ninety healthy school children were used as a control group. The following parameters were investigated: inulin clearance (GFR), urinary excretion of albumin, alpha1-microglobulin (alpha1-MG), calcium, beta-N-acetylglucosaminidase (beta-NAG) and Tamm-Horsfall protein (THP), tubular phosphate reabsorption (TP/Cl(cr)) and percent reabsorption of amino acids (TAA). Significantly lower GFR was found 1 and 2 years after HSCT but within the normal range in the period before HSCT. There was no correlation between GFR within the first month after HSCT and long-term outcome of GFR. Tubular dysfunction was found in 14-45% of patients 1 and 2 years after HSCT depending on the parameter investigated. Pathological values 1 and 2 years after HSCT were found for alpha1-MG excretion in 40% and 39%, respectively, for TP/Cl(cr) in 44% and 45%, for beta-NAG in 26% and 19%. Median TP/Cl(cr) was significantly lower 2 years after HSCT than before. TAA was mildly impaired in 7/14 patients before, in 5/29 one and in 9/29 2 years after HSCT, but median TAA was within normal range at all times. The median excretion of albumin, THP and calcium was within the normal range at all investigations. No influence of ifosfamide pre-treatment on the severity of tubulopathy was found. The investigation of tubular renal function should be part of a long-term follow-up in children after HSCT.