The alizarin red S assay is considered the gold standard for quantification of osteoblast mineralization and is thus widely used among scientists. However, there are several restrictions to this method, e.g., moderate sensitivity makes it difficult to uncover slight but significant effects of potentially clinically relevant substances. Therefore, an adaptation of the staining method is appropriate and might be obtained by increasing the mineralization ability of osteoblasts. In this study, cell culture experiments with human (SaOs-2) and murine (MC3T3-E1) osteoblasts were performed under the addition of increasing concentrations of calcium chloride (1, 2.5, 5, and 10 mM) or calcitonin (1, 2.5, 5, and 10 nM). After three or four weeks, the mineralization matrix was stained with alizarin red S and the concentration was quantified photometrically. Only calcium chloride was able to significantly increase mineralization, and therefore enhanced the sensitivity of the alizarin red S staining in a dose-dependent manner in both osteoblastic cell lines as well as independent of the cell culture well surface area. This cost- and time-efficient optimization enables a more sensitive analysis of potentially clinically relevant substances in future bone research.

Summary: Introduction: As a typical consequence of bleeding into muscles and joints, patients with severe hemophilia suffer from acute and chronic pain. In spite of its high prevalence, pain in this patient group is not always sufficiently considered or treated in an effective manner.Aim: The recommendations presented in this paper address possible improvements in pain management in hemophilia patients and particularities that have to be taken into account in this patient group.Method: The manifold aspects of pain management in hemophilia patients were discussed within the framework of an expert meeting. Based on the available literature and the experts’ clinical experience, the participants developed a set of recommendations presented in this paper.Results: Pain management in patients with hemophilia is often insufficient, a fact that not only influences the patients’ quality of life but also implies the risk of difficult to manage chronic pain. Both the prevalent polypharmacy (due to comorbidities) as well as the underlying disease itself present special challenges to pain therapy in this patient group. The present review and recommendations are intended to support medical professionals in recognising the risks of pain chronicity, applying basic principles of multimodal pain therapy, including the options of psychological intervention and modalities of physical medicine in therapy concepts, and reaching a comprehensive understanding of the range of analgesic options available.

Objective: To review the current knowledge on bone health in patients with hemophilia A and the underlying pathogenetic mechanisms.Data Sources: Original research articles, meta-analyses, and scientific reviews.Data Synthesis: Already in childhood, patients with hemophilia A are prone to low bone mineral density, leading to osteopenia and/or osteoporosis. Initially associated with the life style of hemophilia, today we are faced with accumulating evidence that coagulation factor VIII is involved directly or indirectly in bone physiology.Conclusion: Understanding the role of factor VIII and the mechanisms of decreased bone mineral density in hemophilia A is critically important, especially as non-factor replacement therapies are available, and treatment decisions potentially impact bone health.

Zusammenfassung: Angeborene Störungen der Thrombozytenfunktion sind eine heterogene Gruppe von Erkrankungen, die oft erst bei Auftreten von Blutungen erkannt werden. Im klinischen Bereich haben sich nur wenige Methoden zur Diagnose und Klassifizierung von angeborenen Thrombozytenfunktionsstörungen bewährt. Für eine rationelle Diagnostik ist ein stufenweises Vorgehen empfehlenswert. Anamnese und klinische Untersuchung sind Grundvoraussetzungen. Das von-Willebrand-Syndrom und andere plasmatische Gerinnungsstörungen sollten vor einer spezifischen Thrombozytenfunktionsdiagnostik immer ausgeschlossen werden. Die Bestimmung von Zahl, Größe, Volumen (MPV) und Morphologie der Thrombozyten erlauben Rückschlüsse auf die zu Grunde liegende Störung.Die PFA-100®-Verschlusszeit eignet sich als Screening zum Ausschluss schwerer Thrombozytenfunktionsstörungen. Die Aggrego metrie ermöglicht die Untersuchung zahlreicher Aspekte der Thrombozytenfunktion. Die Durchflusszytometrie ist zur Diagnose von Thrombasthenie Glanzmann, Bernard-Soulier- Syndrom und Freisetzungsstörungen geeignet. Molekulargenetische Untersuchungen können die Verdachtsdiagnose bestätigen oder zum Nachweis nicht beschriebener Defekte verwendet werden. Hier wird die ungekürzte Version der inter -disziplinären Leitlinie* präsentiert.

Summary: The development of neutralizing alloanti-bodies (inhibitors) to factor VIII (FVIII) is one of the most serious complications in the treatment of haemophiliacs. Inhibitors occur in approximately 20 to 30% of previously untreated patients (PUPs), predominantly children, with severe haemophilia A within the first 50 exposure days (ED). Immune tolerance induction (ITI) leads to complete elimination of the inhibitor in up to 80% of the patients and offers the possibility to restore regular FVIII prophylaxis. However, patients with high titre inhibitors, in whom standard ITI fails, usually impose with high morbidity and mortality and therefore prompting physicians to alternate therapy regimens. Rituximab, an anti-CD 20 monoclonal antibody has been successfully used in children and adults for the management of B-cell mediated disorders. We report on the use of a new protocol including rituximab in two adolescents with severe haemophilia A and high titre inhibitors, severe bleeding tendency and high clotting factor consumption after failing standard ITI. Both patients received a concomitant treatment with FVIII according to the Bonn protocol, cyclosporine A and immunoglobulin. Treatment with rituximab resulted in a temporary B-cell depletion leading to the disappearance of the inhibitor. FVIII recovery and half-life turned towards normal ranges. In patient 1 the inhibitor reappeared 14 months after the last rituximab administration. In patient 2 complete immune tolerance could be achieved for 60 months. Bleeding frequency diminished significantly and clinical joint status improved in both patients. In patient 1 the treatment course was complicated by aspergillosis and hepatitis B infection. Conclusion: Rituximab may be favourable for patients with congenital haemophilia, high-titre inhibitors and a severe clinical course in whom standard ITI has failed. Prospective studies are required to determine safety, efficacy and predictors of success.

Hereditary disorders of platelet function are a heterogeneous group of diseases that are often complex and tend to go undetected until clinically relevant bleeding occurs. Hallmarks are epistaxis, easy bruising, mucous membrane bleeding, perioperative bleeding and menorrhagia. Bleeding may be intermittent and unpredictable. After decades of successful research on platelet biology and genetics, research findings have not been satisfactorily translated to clinical practice. The lack of robust and well- standardized test systems continues to make the diagnosis of platelet defects cumbersome for the practising clinician. Patient history and description of clinical bleeding symptoms are essential. Exclusion of von Willebrand disease, platelet count and investigation of blood smears may provide a tentative diagnosis. Light transmission aggregometry is still considered the gold standard for assessing platelet function. Due to the wide range of possible genetic defects molecular biological analyses can complement but do not substitute for other tests. The true incidence of inherited disorders of platelet function is unknown. A survey in Germany revealed that receptor-defects including Glanzmann's thrombasthenia and Bernard-Soulier syndrome and aspirin-like defects were the most frequently diagnosed plartelet disorders. Of affected children 60% presented with mild and 40% with moderate to severe bleeding tendency. Epistaxis, cutaneous and mucous membrane bleeding were the most frequent symptoms. The paediatric competence network of the GTH e.V. comprises 44 collaborating centres that are caregivers to over 150 children with well-defined inherited platelet defects. A major goal of this network is to promote diagnosis of children with inherited disorders of platelet function. Angeborene Erkrankungen der Thrombozytenfunktion sind eine komplexe Gruppe von Erkrankungen, die häufig bis zum Eintritt von Blutungssymptomen unentdeckt bleiben. Typische Symptome sind eine Neigung zu Nasenbluten, Hämatomen, Schleimhautblutungen, perioperativen Blutungen und Menorrhagien. Blutungen treten oft plötzlich und unvorhergesehen auf. Nach Jahrzehnten sehr erfolgreicher Erforschung der biologischen und genetischen Grundlagen der Funktionsstörungen von Thrombozyten fehlt bislang eine zufriedenstellende Umsetzung dieser Ergebnisse in die klinische Praxis. Aufgrund des Fehlens stabiler und standardisierter Testverfahren ist die Diagnose von Thrombozytendefekten auch heute noch sehr schwierig. Die Anamneseerhebung und Beschreibung der Blutungssymptome sind besonders wichtig. Nach Ausschluss eines von Willebrand Syndroms und Beurteilung der Thrombozytenzahl und -morphologie ist häufig bereits eine vorläufige Klassifizierung möglich. Aufgrund der unterschiedlichen genetischen Ursachen kann eine genetische Analyse die Untersuchung der Thrombozyten zwar komplettieren aber nicht ersetzen. Die wahre Inzidenz von angeborenen Thrombozytendefekten ist unbekannt. Eine Unfrage in Deutschland ergab, dass Rezeptordefekte einschließlich der Thrombasthenie Glanzmann und dem Bernard Soulier Syndrom und der Aspirin-like Defekt am häufigsten diagnostiziert wurden. 60% der betroffenen Kinder zeigten eine milde und 40% eine mittelschwere und schwere Blutungsneigung. Epistaxis, Hämatome und Schleimhautblutungen waren die häufigsten Symptome. Das Kompetenznetzwerk der Ständigen Kommission Pädiatrie der GTH umfasst 44 Zentren, die insgesamt über 150 betroffene Kinder mit eindeutig klassifizierten angeborenen Thrombozytopathien betreuen. Ein wichtiges Ziel des Kompetenznetzwerks ist die Förderung der Diagnose von Kindern mit angeborenen Störungen der Thrombozytenfunktion.