[Refractory thrombotic thrombocytopenic purpura]. / Therapierefraktäre thrombotisch-thrombozytopenische Purpura.

Thrombotic thrombocytopenic purpura (TTP) remains a serious illness with potentially life-threatening complications. The following case of a TTP patient describes a serious relapse with exacerbation in spite of adequately initiated therapy and highlights the necessity of interdisciplinary expertise in the treatment of the disease.

Monitoring of complement activation biomarkers and eculizumab in complement-mediated renal disorders.

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.

Large deletions play a minor but essential role in congenital coagulation factor VII and X deficiencies.

UNLABELLED: Congenital factor VII (FVII) and factor X (FX) deficiencies belong to the group of rare bleeding disorders which may occur in separate or combined forms since both the F7 and F10 genes are located in close proximity on the distal long arm of chromosome 13 (13q34). We here present data of 192 consecutive index cases with FVII and/or FX deficiency. 10 novel and 53 recurrent sequence alterations were identified in the F7 gene and 5 novel as well as 11 recurrent in the F10 gene including one homozygous 4.35 kb deletion within F7 (c.64+430_131-6delinsTCGTAA) and three large heterozygous deletions involving both the F7 and F10 genes. One of the latter proved to be cytogenetically visible as a chromosome 13q34 deletion and associated with agenesis of the corpus callosum and psychomotor retardation. CONCLUSIONS: Large deletions play a minor but essential role in the mutational spectrum of the F7 and F10 genes. Copy number analyses (e. g. MLPA) should be considered if sequencing cannot clarify the underlying reason of an observed coagulopathy. Of note, in cases of combined FVII/FX deficiency, a deletion of the two contiguous genes might be part of a larger chromosomal rearrangement.

Pregnancy in Upshaw-Schulman syndrome.

The 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.

Desmopressin testing in haemophilia A patients and carriers: results of a multi centre survey.

INTRODUCTION: 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 Haemorrhagic 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 detected 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.

[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.

Detection of Aspergillus DNA in cerebrospinal fluid from patients with cerebral aspergillosis by a nested PCR assay.

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.

UV-resonance Raman spectroscopic study of human plasma of healthy donors and patients with thrombotic microangiopathy.

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.

Comparison of whole-body STIR-MRI and 99mTc-methylene-diphosphonate scintigraphy in children with suspected multifocal bone lesions.

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 (P<0.001). 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.

[Thrombotic microangiopathy following stem cell transplantation]. / Thrombotische Mikroangiopathie nach Stammzelltransplantation.

Stem cell transplantation associated microangiopathy (TA-TMA) occurs in about 7% of all transplantations. While it resembles in all its features idiopathic TTP or HUS, response to plasma therapy is poor. The overall mortality of patients afflicted with TA-TMA is about 80%. Comparative studies aimed at discovering risk factors for the development of TA-TMA and defined different entities according to clinical, laboratory and outcome parameters. It is thought to be of multifactorial aetiology with the central event of endothelial damage. Intensive chemotherapy and radiation for bmt-preparation, infection in the immunocompromised host and graft versus host disease in conjunction with immunosuppressive therapy i. e. cyclosporine turn the haemostatic balance into a procoagulant state. Therapeutic strategies deduced from idiopathic TTP were disappointing with the only consensus in the withdrawal of cyclosporine from TA-TMA patients. Future trials are directed to ameliorate endothelial dysfunction.

Remission of thrombotic thrombocytopenic purpura in a patient with compound heterozygous deficiency of von Willebrand factor-cleaving protease by infusion of solvent/detergent plasma.

UNLABELLED: Plasma exchange or plasma infusion is considered to be the therapy of choice in patients with thrombotic thrombocytopenic purpura (TTP) who are deficient in von Willebrand factor-cleaving protease (VWF-CP). Recently, mutations in the ADAMTS 13 gene were identified as being responsible for VWF-CP deficiency in patients with familial TTP (VWF-CP deficiency in the absence of an inhibitor). Here we report on a girl who presented with recurrent thrombocytopenia and anaemia since birth, developing the full pentad of characteristic TTP at the age of 16 y. Congenital TTP was confirmed on the basis of severe VWF-CP deficiency in the absence of an acquired inhibitor. The patient was found to be compound heterozygous for two hitherto undescribed mutations in the ADAMTS 13 gene: a truncating frame shift mutation, 4143insA in exon 29, and the nonsense mutation 3100A >T in exon 24 (R1034X). After infusion of solvent/detergent plasma, the patient went into remission and remained asymptomatic under regular plasma therapy at 2-wk intervals for over two years. CONCLUSION: TTP in childhood may be mild and oligosymptomatic. Determination of VWF-CP activity is helpful in the differential diagnosis of thrombocytopenia.

Renal function in long-term survivors of stem cell transplantation in childhood. A prospective trial.

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.