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Acquired haemophilia A (AH) is a rare bleeding disorder caused by an auto-antibody to coagulation factor VIII. It is associated with various autoimmune diseases, pregnancy, cancer or drug ingestion; however, in 50% of patients, no underlying disorder is found. In the present study, we investigated the association of HLA class I (A, B and Cw) and class II (DRB1 and DQB1) alleles with AH in a cohort of 57 patients. While no association with any class I allele was detected, a significantly higher frequency of DRB1*16 [odds ratio (OR) 10.2, 95%CI: 5.32-19.57, P < 0.0001] and DQB1*0502 (OR 2.2, 95%CI: 1.12-4.54, P < 0.05) was observed. In contrast, the frequency of DRB1*15 and DQB1*0602 alleles was found to be decreased in patients with AH corresponding to an OR of 0.4 for both HLA loci. Upon comparing the frequencies of these alleles with those of patients with congenital haemophilia A with inhibitors, the data demonstrate that the high risk alleles in patients with AH DRB1*16 and DQB1*0502 are found to be low risk alleles in patients with congenital haemophilia A with inhibitors (OR 1.1 and 1.5 respectively). Conversely, the alleles that exhibit low risk in AH DRB1*15 and DQB1*0602 are found to be high risk for haemophilia A inhibitor patients (OR 2.2 and 3.7 respectively). The pathophysiological reason for this finding remains unknown. It might be speculated that the presence or absence of the FVIII antigen and the various ability of HLA molecules to present the FVIII antigen to the T-cell receptor contribute to these findings.

ZusammenfassungDie Hämophiliekommission der GTH betreibt ein zentrales Register für alle deutschen Zentren, die Patienten mit Blutungsneigungen behandeln. Das Ziel war ein System, mit dem epidemiologische Daten bzgl. Gerinnungsstörungen effektiver gesammelt und ausgewertet werden können. Das Register bildet außerdem die datentechnische Grundlage im Rahmen des Deutschen Human-Genom-Projekts. Erklärtes Ziel ist dabei die vollständige molekulare Charakterisierung der genetischen Mutationen des X-Chromosoms von Patienten mit Hämophilie A in Deutschland und die anschließende Korrelation mit dem Phänotyp. Ein elektronisches Netzwerk wird für die Kommunikation benutzt. Eine Java-Applikation wurde entwickelt, wodurch die Online-Datenerfassung durch die teilnehmenden Zentren möglich wurde. Alternativ wurde die Möglichkeit zur Offline-Datenerfassung mit Versendung von verschlüsselten Datenträgern geschaffen. Der Datenschutz ist durch personenbezogenen Zugangsschutz, Anonymisierung der Patientendaten und Kodierung der Daten mit einem Verschlüsselungsverfahren gewährleistet. Die Bestätigung der Datenschutzbehörden über die Unbedenklichkeit des Projekts liegt vor. Insgesamt wird durch das Hämophileregister eine Verbesserung der epidemiologischen Wissensbasis als Grundlage für zukünftige therapeutische Entscheidungen bzgl. Grund- und Begleiterkrankungen erwartet. Das Register kann außerdem weiteren Forschungsprojekten als Basis dienen.

ZusammenfassungEtwa 30% der Patienten die an schwerer Hämophilie A leiden, entwickeln als Komplikation einer Substitutionstherapie gegen Faktor VIII gerichtete Antikörper, die die prokoagulatorische Aktivität von intravenös injizierten FVIII-Proteinen neutralisieren. In der Regel werden verschiedene Epitope auf dem FVIII-Molekül durch diese Antikörper gebunden. Die detaillierte Struktur dieser Epitope ist unbekannt. In dieser Studie werden Epitope auf dem Faktor-VIII-Molekül mit Hilfe festphasengebundener Peptid-Arrays identifiziert, auf denen die Aminosäuresequenz des FVIII in Oligopeptiden dargestellt wird. Die Bindung von FVIII-Antikörpern an bestimmte Peptidsequenzen zeigt potenzielle Epitopstrukturen. FVIII-Antikörper von Inhibitorpatienten und gesunden Blutspendern werden zurzeit mit dieser Methode charakterisiert. Identifizierte Epitope können zur Entwicklung neuer Therapiekonzepte führen, die dazu dienen, eine Hemmkörperentwicklung zu vermeiden bzw. bereits entstandene Hemmkörper besser zu eliminieren. Als Teilprojekt des Konsortiums »Hämophilie A« wird im Rahmen des Gesamtvorhabens »Genotyp-Phänotyp-Korrelation bei der Hämophilie A« untersucht, ob die Epitopcharakteristik mit dem Typ und der Position der FVIII-Genmutationen korreliert sowie Zusammenhänge zwischen Epitop und klinischem Verlauf der Krankheit bestehen. Zusätzlich wird der Einfluss der Epitope auf den therapeutischem Effekt und Erfolg der Immuntoleranzinduktion untersucht.

ZusammenfassungDie Hämophilie A wird durch einen genetischen Defekt des FVIII-Gens verursacht und führt entweder zum vollständigen Fehlen oder zu einem erheblichen Funktionsverlust des Gerinnungsfaktors VIII im Blut. Die erhöhte Blutungsneigung der betroffenen Patienten kann durch die intravenöse Gabe von Faktor-VIII-Protein verhindert werden. Allerdings entwickeln 25% der Patienten, die an einer schweren Hämophilie A leiden, Anti-Faktor-VIII-Antikörper gegen das substitutierte Konzentrat. In dieser Studie wird versucht, die phänotypischen Parameter (z.B. detaillierte Dokumentation des Krankheitsverlaufs, Basislaborwerte) und therapieassoziierten Daten (z.B. verwendetes Konzentrat, Konzentratverbrauch, Anzahl der Substitutionen, Faktor-VIII-Recovery sowie Inhibitorentwicklung und -eliminierung) von etwa 800 Patienten zu erfassen. Durch diese Erfassung kann der Effekt der unterschiedlichen Therapiemaßnahmen auf den Krankheitsverlauf bewertet werden. Einen besonderen Einfluss auf den Krankheitsverlauf und die Therapie hat der Faktor-VIII-Gendefekt, der ursächlich für die Hämophilie verantwortlich ist. Wir erwarten, dass bestimmte Mutationen bzw. Mutationstypen spezifischen Phänotypen und in etwa vergleichbaren therapeutischen Verläufen zugeordnet werden können.

ZusammenfassungDie Hämophilie ist die häufigste genetisch bedingte Form einer schweren Blutungsneigung. Ursächlich für die Erkrankung sind Defekte im Faktor-VIII-Gen, die zu verminderter Faktor-VIII-Aktivität mit einer vom Ausmaß der Störung abhängigen Blutungsneigung führen. Die ursächlichen Gendefekte können inzwischen routinemäßig identifiziert werden. Trotz der Vielfalt der Mutationen lassen sich mehrere Hotspots feststellen, z.B. Inversion im Intron 22, kleine Deletionen/Insertionen an Poly-A und Punktmutationen an CpG-Dinukleotiden. Bereiche geringer Mutationshäufigkeit sind z.B. im mittleren Abschnitt des Exons 14, der für die funktionell wenig bedeutende B-Domäne kodiert und praktisch keine Missense-Mutationen enthält. Die Mutationsdiagnostik verbesserte die Diagnostik und das Verständnis der Pathogenese der Hämophilie-A-Erkrankung. Nach Identifizierung des Gendefekts in einer Familie ist eine schnelle und sichere Konduktorinnendiagnostik möglich. Die Korrelation zwischen Gendefekt und klinischem Verlauf zeigte, dass die Art der Mutation einen wichtigen genetischen Prädispositionsfaktor für die zurzeit schwerste therapeutische Komplikation bei Hämophilie, die Hemmkörperbildung, darstellt. Ein relativ leichter Verlauf bei eigentlich schwerer Hämophilie A lässt sich mit speziellen Mutationen oder gleichzeitig vorliegenden thrombophiliefördernden Mutationen erklären. Molekulare Modelle des Faktor-VIII-Moleküls erlauben gezielt Auswirkungen von Mutationen zu untersuchen und so neue Struktur/ Funktionsbeziehungen zu erkennen. Dies könnte die Entwicklung zukünftiger rekombinanter Gerinnungspräparate mit veränderten Eigenschaften (z.B. höherer Wirkungsgrad, verlängerte Halbwertszeit, geringere Immunogenität) anstoßen.

SummaryTo reduce the risk of transmission of hepatitis A virus, an Octaphar-ma produced factor VIII (fVIII) concentrate treated with solvent detergent (FVIII-SD) was further pasteurized after purification. This product, Octavi SDPlus (FVIII-SDP), was marketed in Europe in 1993 to 1995. Inhibitors appeared from September to October, 1995, in 12 of 109 previously treated German hemophilia A patients. A study of similarly treated Belgian patients, who also developed inhibitors, had shown antibodies to the fVIII light chain (domains A3-C1-C2) only. In the present study, the epitope specificity of 8 German inhibitor plasmas was also found to be restricted to the light chain. In radioimmunoprecipitation assays to localize the light chain epitope(s), antibody binding to heavy chain (domains A1-A2-B) was 11-148 fold lower than to the C2 domain, and binding to recombinant A3-C1 was barely detectable. These results were supported by >95% neutralization of a high responder inhibitor titer by the C2 domain.

Conventional visual analysis of the EEG was performed on 320 hemophiliacs infected with HIV, who spanned the range of the Walter Reed (WR) system for classifying clinical stage of HIV infection, and on 50 HIV seronegative hemophiliac controls. Intermittent or paroxysmal slowing was the conventional EEG abnormality most commonly seen in early stages of HIV infection (stages WR1 and 2), with increased focal epileptiform activity and generalized slowing appearing in patients with the full clinical syndrome of AIDS (WR6). Slowing of the manually measured alpha rhythm was noted in stages WR2 and above. Quantitative EEG (qEEG) was obtained in a subset of 103 male HIV seropositive male hemophiliacs and 35 male HIV seronegative hemophiliac controls. The principal findings were a progressive relative increase in theta power with a tendency towards an anterior topographic distribution, and a progressive decline of spectral power in fast alpha relative to slow alpha with increasing severity of HIV disease. Significant qEEG differences from controls were apparent in WR2 subjects (seropositive with lymphadenopathy and without other constitutional symptoms), and were relatively greater in WR3-6 subjects. These results suggest sensitivity of qEEG to early CNS involvement with HIV infection.

Summary An immunoaffinity purified, solvent/detergent virally inactivated factor VIII (FVIII) concentrate (Hemofil® M) has been in clinical use in persons with haemophilia A since March 1987 and under clinical trial prior to licencing in the USA, Europe and Japan. The specification set and consistently met for the monoclonal antibody (mAb) content of the final product is 0.1 ng/IU FVIII. This level was considered non-immunogenic, based upon animal studies, recommendations of the European PHarmacopoeia with ovalbumin contamination of influenza vaccine and also the experience during clinical research studies using aAb for diagnostic or other clinical purpose as reported in the medical literature. This research communication documents the surveillance for human anti-murine antibodies (HAMA) in 13 patients with serve haemophilia A. These patients have been treated with a large amount of product (mean 14,026 IU), numerous different lots of Hemofil M (total 45; mean 17) over a substantial period of time (mean 28 months). The data generated from this group of 13 patients before and during therapeutic Hemofil M administration failed to show the development of any HAMA response at any timepoint. Therefore Hemofil M prepared by mAb resin immunoaffinity column chromatography under the currently set specifications for mAb contaminant of 0.1 ng/IU FVIII or less is a safe therapeutic agent in the management and prevention of bleeding episodes in persons with haemophilia A.

The incidence of male-to-female transmission of HIV infection was studied in a population of 198 sexual partners of hemophiliacs who tested HIV positive since 1984. The follow-up observation period was 1987–1992. Transmission occurred in 20 (10%) cases. The analysis of risk factors for transmission was performed in a subgroup of 57 hemophiliacs with seronegative sexual partners as compared to eight transmitters. Transmitters showed a significantly more advanced immune depletion at enrollment as well as at the end of the observation period. Furthermore, transmitters had a more advanced disease at the end of the study (75% vs. 29% CDC IV; p

SummaryThe formation of factor VIII antibodies is a major problem for replacement therapy of haemophilia A patients. Antibodies occur in 5-30% of patients with severe haemophilia A. The reason for antibody formation is still unknown. In this study we correlate for the first time different factor VIII gene mutations, stop- and missense mutations, large and small deletions and intrachromosomal intron 22 recombinations to antibody formation. A total of 364 patients with known inhibitor status of our institute, of the database, and of 3 studies representing intron-22-inversion data are included. The results show that the risk for developing factor VIII antibodies is strongly related to stop mutations, large deletions and intrachromosomal recombinations. A probable explanation could be the complete lack of endogenous circulating factor VIII protein in these cases. Other factors that might be important for the pathogenesis of inhibitor formation, e. g. the antenatal period, as well as possible therapeutic effects, are discussed.

SummaryThe pharmacokinetics and tolerability of factor XIII (FXIII) from plasma were compared with those of FXIII from placenta in a randomised, double-blind, crossover study involving 13 patients with congenital FXIII deficiency. Both FXIII activity and FXIII antigen were monitored. No difference was seen in the mean half-lives of the two preparations (9.3 days and 9.1 days for plasma and placenta FXIII activity, respectively). Response was similar for both preparations, but was slightly greater for FXIII from plasma.Similar results were found for recovery (65% vs 60%). The area under the data completed by extrapolation was significantly higher for FXIII from plasma. No differences between preparations in terms of efficacy or tolerability were observed. It can be concluded that treatment with FXIII concentrate from plasma is as efficient as with FXIII concentrate from placenta in terms of recovery and half-life. Both preparations were equivalent in terms of safety during the observation period. With the administration of monthly injections of approximately 30 U/kg serious bleeding events were prevented and no other serious adverse events occurred.

The Bonn Haemophilia Care Center provides patient care on a superregional level. The centre's large service area is, in part, due to the introduction of haemophilia home treatment and related to this the individualized prophylaxis in children and adults by Egli and Brackmann in Bonn in the early 1970s, that represented a milestone in German haemophilia therapy. Epidemiologic patient data from the two selected time points, 1980 and 2009, are evaluated to illustrate the change in the composition of the patient clientele. In 1980 a total of 639 patients were treated at the Bonn Haemophilia Center. 529 patients exhibited a severe form and 110 a non-severe form of the respective clotting disorder. In 2009 the Bonn Haemophilia Center took care for a total of 837 patients. There were 445 patients who suffered from a severe form of the considered clotting disorder while 392 showed a non-severe course. The number of less severely affected patients has increased significantly in 2009. Patients in 1980 were predominantly suffering from a severe form and most had to travel more than 150 km from their homes to the treatment center. In 2009 the number of patients living a medium-long distance from the care provider has significantly increased while the number of patients living more than 150 km from the center has decreased. Comparing 2009 to 1980 a growth of the center's regional character becomes apparent, especially when patient age and severity of the coagulation disorder are taken into consideration. The regional character was more strongly pronounced with milder disease severity and lower patient age. Due to the existence of well established primary haemophilia care in CCCs in Germany, the trend for the recent years is that the proportion of young patients that choose haemophilia care providers closer to their homes is increasing.

In 1990, 7 hemophilia B patients were infected with human immunodeficiency virus type 1 (HIV-1) after exposure to a single common lot of clotting factor. The hypervariable regions V1 and V2 of the proviral env gene from the patients shared a homology between 97.5% and 100% at the time of seroconversion. To determine the in vivo diversification of these epidemiologically closely related virus strains, the patients were followed up in the early phase of HIV infection. Direct sequencing of the V1/V2 region in the env gene still revealed a very high degree of homology (96.5%-100%). In the case of the patient who showed the highest decrease of CD4+ cells, moderate genetic diversification of the virus was associated with a biological differentiation. The strain originally presenting two expressed substitutions displayed three more deviations 9 months after the first investigation (including one reversion to the consensus sequence). In addition, the virus that originally could not be cultivated could now be isolated as a low cytopathogenic agent. This study provides evidence that the high genetic homogeneity of HIV-1 observed at the time of seroconversion is maintained as a predominant consensus sequence in the following so-called latent phase of infection.

The frequency, presentation, diagnosis and clinical course of Pneumocystis carinii infections (PCI) were studied during aerosolized pentamidine prophylaxis (AP) and its impact on the spectrum of AIDS-related and other pulmonary infections in HIV-infected hemophiliacs. We conducted an open study on primary (PP) and secondary (SP) AP. Breakthrough P. carinii infections (BPCI) and other infectious complications were analyzed retrospectively. Hemophiliacs without prior P. carinii pneumonia (PCP) who had been reluctant to any prophylaxis and who developed PCP served as control group. Statistical analysis of the efficacy of prophylaxis was performed by calculating confidence intervals of binomial p. Of 73 hemophiliacs (56 on PP and 17 on SP) 10 developed BPCI (7 in PP and 3 in SP) during a mean observation time of 14.9 months (range 0.5-30); total 13.6% (6.7%; 23.7%), PP 12.5% (5.1%; 24%), SP 17.6% (3.7%; 43.4%), confidence intervals at a level of 95%. Three BPCI presented atypically with cavitation (1), pneumothorax (2), Pneumocystis pleuritis (1), dissemination (2) as compared to none in the control group. Sensitivity of bronchoalveolar lavage (BAL) was 88.9%, specificity 100% (both 100% in the control group). PCP was the leading AIDS manifestation (21.3%), CNS manifestations taken together were more frequent (36.2%). Bacterial pneumonia was the most frequent respiratory infection. One patient of the study group with recurrent pneumothorax possibly died of BPCI as compared to no BPCI-related deaths in the control group. Efficacy of prophylaxis in hemophiliacs was comparable to other risk groups. AP alone may be insufficient for the control of PCI in patients with long-term profound immunodeficiency, especially in SP. 30% of BPCI presented atypically.(ABSTRACT TRUNCATED AT 250 WORDS)

Acquired haemophilia A (AH) is a rare bleeding disorder caused by an auto-antibody to coagulation factor VIII. It is associated with various autoimmune diseases, pregnancy, cancer or drug ingestion; however, in 50% of patients, no underlying disorder is found. In the present study, we investigated the association of HLA class I (A, B and Cw) and class II (DRB1 and DQB1) alleles with AH in a cohort of 57 patients. While no association with any class I allele was detected, a significantly higher frequency of DRB1*16 [odds ratio (OR) 10.2, 95%CI: 5.32-19.57, P0.0001] and DQB1*0502 (OR 2.2, 95%CI: 1.12-4.54, P0.05) was observed. In contrast, the frequency of DRB1*15 and DQB1*0602 alleles was found to be decreased in patients with AH corresponding to an OR of 0.4 for both HLA loci. Upon comparing the frequencies of these alleles with those of patients with congenital haemophilia A with inhibitors, the data demonstrate that the high risk alleles in patients with AH DRB1*16 and DQB1*0502 are found to be low risk alleles in patients with congenital haemophilia A with inhibitors (OR 1.1 and 1.5 respectively). Conversely, the alleles that exhibit low risk in AH DRB1*15 and DQB1*0602 are found to be high risk for haemophilia A inhibitor patients (OR 2.2 and 3.7 respectively). The pathophysiological reason for this finding remains unknown. It might be speculated that the presence or absence of the FVIII antigen and the various ability of HLA molecules to present the FVIII antigen to the T-cell receptor contribute to these findings.

A total of 181 human immunodeficiency virus (HIV)-seropositive hemophiliacs and 28 hemophilic controls were evaluated by psychometric tests and by electroencephalogram (EEG). Patients were classified from stages 1-6 according to the immunological criteria of the Walter Reed staging system. Statistical analysis of psychometric data showed an effect of the stage of the disease on test performances, indicating a decline in attention, accumulation of perceptual interferences, decline in visuoperceptual speed and visuomotor response speed and reduced verbal memory performance, especially in stage 6 patients. Comparison of performance levels with normative test data already revealed cognitive deficits in about 20-30% of the patients in stages 2-5. As regards verbal memory, especially learning and recognition of new verbal information were impaired. In contrast, there was no significant deficit for nonverbal memory processing. Compared with the controls, patients exhibited an increasing number of abnormal EEG findings in stages 2-6. As a conclusion, in the stages before acquired immunodeficiency syndrome (AIDS) develops, EEG findings as well as psychometric findings indicate central nervous system involvement of AIDS in about 20-30% of cases, whereas in full-blown AIDS there is a marked increase to 80%. Besides a more general deficit of attention and psychomotor speed as is seen in subcortical dementia, there is evidence for a particular verbal learning disorder, suggesting additional selective impairment of the brain.

Between 1978 and 1989, a controlled study was performed on children and adolescents suffering from severe haemophilia A. Special attention was given to long-term therapy adjusted to the patients’ requirements. The clinical and the X-ray scores of the Orthopaedic Advisory Committee of the World Haemophilia Society were used to assess the orthopaedic findings. The present investigation is a 12-year follow-up study of 90 patients affected with severe forms of haemophilia A. All knee and ankle joints of the patients were evaluated. After 12 years, the clinical scores remained unchanged or had been improved (knees 94%, X-ray score 97, ankles 86, X-ray score 88).

Acquired haemophilia (AH), an autoimmune disorder with clinical features ranging from harmless haematomas to life-threatening bleedings, still has a mortality rate of up to 25%. Owing to its low frequency (1-4 x 10(6)), standardized treatment protocols for its variable manifestations are not available. In case of prominent severe bleedings, the treatment should aim at rapid elimination of the antibody to protect patients from bleedings and on reinduction of long-term immune tolerance. Clinical data, short- and long-term treatment results of 67 patients diagnosed by our centre are presented. Patients were treated depending on their bleeding severity either by an immunosuppressive treatment alone, or in case of life-threatening bleedings, by a combined protocol (modified Bonn-Malmö protocol, MBMP) consisting of antibody depletion through immunoadsorption, intravenous immunoglobulin treatment, immunosuppression and high-dose factor VIII (FVIII) substitution. Mild bleedings occurred in two patients who were treated successfully alone by immunosuppression. Complete remission (CR) was achieved in 90% of the patients treated with MBMP (60). Of the six patients (10%) who achieved a partial remission (PR), four suffered from cancer. Mortality under MBMP was not seen. In contrast, five patients, in whom diagnosis of AH was delayed, experienced fatal outcome during surgical interventions before initiation of MBMP treatment. Prognosis in AH depends mainly on its prompt diagnosis. Treatment procedures should be adapted to bleeding severity and inhibitor titres. Under these conditions, AH is a potentially curable autoimmune disorder with an excellent prognosis.

In hemophilia A, determination of factor VIII activity is used to classify the extent of impaired hemostasis and to estimate bleeding risks of patients. In contrast, the now established assays of thrombin generation (TGA) (1, 2) are not based on a single clotting factor, but are reflecting the action of the total clotting mechanism. Especially in hemophilic patients, TGA offers the possibility to monitor the treatment with haemostatic agents.

This case report demonstrates that, in selected hemophilic patients, simultaneous bilateral knee replacement surgery can be performed with a good outcome without a significant increase in perioperative risk.The overall costs, especially for the FVIII replacement, are significantly lower in the simultaneous bilateral surgery than in the unilateral staged surgery. The authors continue to offer hemophilic patients the option of simultaneous bilateral total knee arthroplasties.

Adequate factor substitution is a prerequisite for successful conservative or operative treatment of haemophilic synovitis. Subsequent prophylactic factor substitution can prevent further joint problems in the future. Orthopaedic treatment regimes are dependent on the classification of the synovitis and the stage of the arthropathy. Synovitis can be classified into acute and chronic forms. The arthropathy is classified according to Arnold and Hilgartner. When the synovitis has already become chronic, early arthroscopic synovectomy is recommended in order to prevent the otherwise inexorable progress of the arthropathy. Failure to treat in the early phase of the pathology causes problems in the correction of further stages. The progression of the joint disease can be shown both clinically and radiologically as the arthropathy develops, and influences the treatment.

Congenital factor XIII (FXIII) deficiency (OMIM: +134570, +134580) is known as a rare autosomal recessive disorder, affecting one in 1–3 million individuals. Deleterious mutations occurring in F13A gene or rarely (< 3% of all cases) in F13B gene result in absence or reduction of plasma FXIII activity. Patients suffering from this condition are characterized by life-long bleeding diathesis and/or impaired wound healing, and spontaneous abortions in affected females as well. The F13A gene maps to the short arm of chromosome 6 (p24–25) and spans about 160 kb of genomic DNA. It consists of 15 exons encoding a mature protein of 731 amino acids. The F13B gene is located on the long arm of chromosome 1 (q32–32.1) and contains 12 exons encoding the mature protein of 641 amino acids [1, 2].

Hemophilia A (HA) is caused by impaired F8 protein activity. Various point mutations leading to either altered or truncated FVIII protein and the two major inversion hot spots, intron 22 and intron 1 inversions, have been reported. Most interestingly, in about 2% of the HA cases we were unable to detect any mutation(s), even after sequencing the whole F8 exons and their flanking intronic sequences [1]. Therefore, to further investigate the reason for HA in these small number of patients we have recently analyzed the F8 mRNA, isolated from lymphocytes, without finding any rearrangements [2]. However in one severe HA patient, we were unable to detect a RT-PCR product from the F8 gene. We followed the segregation of this allele in the family members, mainly his mother and his sister; the same allele present in the patient was found to be transmitted by the mother to his sister. In all three individuals we were unable to get an RT-PCR product corresponding to this allele (Fig. 1). Our data strongly suggest that the cause of HA in this patient is either absence or rapid degradation of the F8 mRNA, thus pointing to a novel mechanism leading to hemophilia A.

Motion analysis was performed in 203 children with hemophilia in a prospective multicenter trial involving German hemophilia centers in Hamburg, Bremen, Hanover, Brunswick, Bonn, Frankfurt, Wurzburg, Ulm, Berlin, Potsdam, Magdeburg, Halle, Erfurt, Erlangen and Munich. 406 knees were investigated in total. Full data sets are available for 172 children to date. The children were aged 3 to 18 and the average age was 9.75 years. Both knees were analyzed during treadmill walking and during knee bends. Motion analysis was conducted using an original ultrasound topometer. Two transmitters were attached above and below the knee joint for this purpose. Threedimensional spatial data were determined accurate to the millimeter and converted mathematically to angle, angular velocity, angular acceleration, and the roll-andglide pattern during knee bends. In addition, a hematology questionnaire, sport and activity questionnaire, and pain questionnaire were completed and an orthopedic examination was performed. The motion analysis data were rated according to a score which factored in regularity, rhythm and sinus curve pattern. Analysis was continued on the basis of these score data. Correlations between epidemiology data and function score Gait pattern displayed a relative dependence on age, with a regression coefficient of 0.56. Knee bends display an even closer correlation with age, with a regression coefficient of 0.59. In contrast, roll-and-glide pattern in the knee joint displays no correlation with age, with a coefficient of 0.04. Both height and weight display a similar dependency as age but are less robust as a basis for drawing conclusions. Interesting results were obtained in terms of a correlation between extent of sporting activity and the respective biomechanical score. Nine children practiced no sports, 73 children practiced sports for up to one hour per week, 65 children for up to 2 hours, and 33 children for more than 2 hours per week. The extent of sporting activity had no effect on the gait score. Knee bend analysis showed, however, that sporting activity was associated with a higher knee bend score. This is merely a trend which was identified. Statistical significance has not been achieved. In particular, the 9 children who did not practice

Alloantibodies (inhibitors) against factor VIII (FVIII) in Hemophilia A represent a major complication in patient care because they render classical substitution therapy ineffected. Inhibitors occur at a frequency of 20-30 %. Herein we report on 432 patients with severe hemophilia A, known mutation with the FVIII gene and known inhibitor status representing the largest cohort ever been reported from a single center (Bonn). The underlying mutation was correlated to the inhibitor prevalence as well as to the inhibitor titer. From the 432 patients, 205 (47,45 %) hemophiliacs showed the prevalent intron22-inversion, 8 (1.85 %) the intron-1-inversion, 61 (14.12 %) a nonsense mutation, 65 (15.05%) a small deletion/insertion, 61 (14.12%) a missense mutation, 17 (3.94%) a large deletion and 15 (3.47 %) a splice site mutation. 79 (18.29 %) of the 432 patients developed an inhibitor of which 46 (58 %) showed a high titer and 33 (42 %) a low titer. Inhibitor prevalences for the main mutation types were 35.29 % in large deletions, 25 % in intron-1-inversion, 21.46 % in intron-22-inversion, 21.31 % in nonsense mutations, 13.85 % in small deletions/insertions, 13.33 % in splice site mutations and 4.92 % in missense mutations. However, for mutations subtypes inhibitor prevalences could be as high as 100 % (3 out of 3) in multi domain large deletions and as low as 0 % (0 our of 16) in small deletions/insertion affecting an adenine series. Although for some mutation types the numbers are still small the results corresponding well to those reported earlier on merged patient samples and underline the significant influence of the mutation type to the risk of inhibitor formation.

Severe hemophilia A (FVIII rest-activity < 1%) was diagnosed post partum due to positive family history. At the age of eight months, first FVIII therapy was given to stop a mucosal bleed. Other FVIII substitutions followed on demand. In September 1989, at the age of two years, he developed a high-titer inhibitor. An immune tolerance therapy (ITT) was started with 180 IU/kg FVIII (plasma derived (pd), containing VWF) twice daily according to the »Bonn Protocol«. The inhibitor first increased to 16 Bethesda units/ml (BU/ml) and decreased afterwards to 1 BU/ml. FVIIIactivity was detectable whereas the FVIII-recovery did not normalize. FVIII administration got more difficult, a central venous line was implanted. The course of therapy was complicated by several infections of the central venous line, thrombosis, sepsis and severe joint bleeds. The central venous line had to be taken out twice; in addition, compliance was difficult. Therapy was not given continuously. The inhibitor titer ranged between 1 and 88 BU/ml over several years. Since severe bleeds could not be avoided, therapy was continued with aPCC twice daily in addition to FVIII. After six years of ITT and still a high-titer inhibitor, the FVIII and

Ten subjects with severe hemophilia and severe hemarthropathy of both knee joints were treated for three months with a magnetic field applicator (manufacturer: Schober). The patients performed no other physiotherapy during this period. Their prior pain management regimen and factor VIII supply were continued unchanged. Motion analysis on a treadmill and during knee bends was performed for both knees before and after starting magnetic field therapy. Patients were provided with the Schober Medicare GmbH Enzymed 5004 magnetic field system consisting of a controlling device, plug and an applicator worn as a cuff on the extremities. The system produces a pulsating magnetic field, primarily in an oscillation range of 2 to 22 Hz. The impulse is a 5-fold sawtooth ramp signal. The treatment period per application is 20 minutes. The device is programmed to automatically produce a gradually beginning and gradually ceasing magnetic field effect at a range of 40 to 90 microtesla. Volunteers use the system 12 times daily on the affected knee joints for a period of three months.

Autoantibodies directed against clotting factors can induce life threatening bleeding with a mortality rate up to 22%. Although the incidence of the disease is low (1-4 x 10(-6)), costs of treatment due to long-term clotting factor substitution can be enormous. Aim of an optimal treatment strategy should be to control bleedings by a rapid and safe elimination of the inhibitor and reinducing long-term immune tolerance.Treatment of 48 patients with acquired haemophilia A (m=20, f =28, age 61.3 (SD 16.4)), the largest patient collective world-wide, was monitored for a mean of 48 months. Three patients received only conservative treatment. 45 patients were treated intensively by a multimodal strategy including: 1. immunoadsorption for antibody elimination; 2. FVIII substitution; 3. intravenous immunoglobulin substitution and 4. immunosuppression. The times required for inhibitor elimination, factor VIII substitution and the duration of the MBMP were documented.In 45 patients with a high titre critical bleeding was controlled immediately after the initiation of MBMP. There were no deaths from bleeding or the treatment. Inhibitor levels decreased to undetectable levels within a median of 3 days (95% CI, 3-7 days), factor substitution was terminated within a median of 13 days (95% CI, 10-16 days) and the treatment was completed within a median of 15 days (95% CI, 13-17 days). The overall response rate for complete remission (CR) was 91%. When cancer patients were excluded, the CR rate was 97%.Considering the short duration and amount of factor VIII substitution, the short time of hospitalization and the long-term median follow up of 48 months without bleeding events, the MBMP appears to have a modifying effect on the immunological response.

101 youngsters aged 8 to 25 (mean 13.3 years) underwent a 5-stage fitness check at six different investigational sites. The 5-stage fitness check comprises a coordination measurement element measuring the excursion of a two-dimensional mobile floor platform (Posturomed) in millimeters over a period of 30 seconds with the subject standing on one leg. Flexibility testing analyzes chest muscles and ischiocrural (hamstring) muscle using an electronic inclinometer. Muscle strength is measured by isometry of the abdominal and back muscles, for each set of muscles on its own and in relation to the other. The endurance test is done on an exercise bike. Resting heart rate and frequency of training are entered as baseline parameters. This test is based on validated IPN endurance testing which produces an individual result in four to six minutes. Test element 5 is infrared body fat analysis on the anterior biceps of the dominant hand. This is the site with the best correlation to hydrostatic weighing. All tests are normalized on the basis of age, gender, height, weight, our own parallel measurements and height and weight percentiles taken from the literature. The results are rated by comparing the specific readings with the relevant agematched means. Scores are given on a scale of 1 to 5.

I’m very pleased to start this conference on’ hemophilia and sport” with my report about the importance of rehabilitation in connection with complete care of hemophiliacs. Rehabilitation measures often have a primary prophylactic function when treating these patients.

Here on we report on the detailed RNA analysis of the factor VIII cDNA from patients that have either putative splicing site mutations or patients with no previously detected DNA mutations by mutation screening and also sequencing of the complete factor VIII cDNA. This protocol proofs to be effective, simple and able to detect splicing errors, thus showing evidence for the causality of splicing defects for the hemophilia A disease in those individuals. Patients that were negative for mutation detection protocols and also show a normal mRNA might be potential candidates for the discovery of novel allelic or non allelic mutations leading to a hemophilia A phenotype.

A generation of hemophiliacs with no bleeding-related impairment of the locomotor system has grown up in Germany over the past two decades. This happy circumstance is the result of preventive treatment of the underlying bleeding disorder and immediate curative treatment of any bleeding on the basis of ready access to sufficient quantities of coagulation factor concentrates. With no restrictions on their freedom of movement, young hemophiliacs of this generation feel free to exercise activities which in the past would have been totally inconceivable.

Today’s generation of hemophiliac children benefits from adequate substitution and radiologically and clinically healthy joints. With self-confidence on their side, it is natural for these young people to want to take up sports. This natural desire gives rise to a number of questions, such as

The aim of this study was to report on the long-term follow-up of haemophilic children with avascular femoral head necrosis and to determine whether radiographic findings at initial diagnosis have any prognostic value. Seven patients with avascular necrosis of the femoral head were clinically and radiographically observed over a period of 5-50 years. The average age of patients at first diagnosis was 7.1 years. At follow-up, three of seven patients claimed to have occasional mild pain in the affected hip, four of seven showed loss of range of motion in the hip joint and two of seven patients showed a limp. Only one patient was clinically completely inconspicuous. The radiographically measured caput-collum-diaphysis angle at follow-up was pathologic in four cases and in one case a lateral subluxation of the femoral head was found. There was marked deformation of the femoral head in three of seven cases and a further two hips showing slight incongruency. Owing to the small patient-number, a statement concerning the prognostic value of defined radiographic signs cannot be made. As expected, the more 'risk signs' radiographically found, the higher the likelihood that patients will suffer arthrosis at a later stage. We propose that a clear distinction between haemophilic arthropathy of the hip and Legg-Calvé-Perthes disease should be made. In cases where radiographic changes are also found in the vicinity of the acetabulum, it is indicative for haemophilic arthropathy.

In this study the long-term value of corrective osteotomy around the knee was evaluated by means of clinical and radiographic parameters. Between 1974 and 1984 we performed 52 corrective osteotomies in the vicinity of the knee on patients affected by haemophilic arthropathy. Forty-two patients (45 osteotomies) were adequately followed-up at an average 11.6 years postoperatively. Using the clinical score of the Advisory Committee of the World Federation of Haemophilia, 38 patients showed a postoperative improvement, five remained clinically unchanged and two showed deterioration. Range of motion of the knee joint did not significantly improve postoperatively. The radiographic Pettersson score showed only a marginal decrease by an average 0.003 points at the time of follow-up. Only one patient needed subsequent joint replacement of both knees, on the left side 13 years after osteotomy and on the right side 8 years after osteotomy. Even in cases of marked radiographic joint destruction, corrective osteotomy shows acceptable long-term clinical results, underlining the feasibility of this management option in the treatment of haemophilic arthropathy of the knee. Although moderate cartilage degenerations in the femoropatellar complex and in the contralateral compartment can be tolerated, this therapy should primarily be contemplated for those patients where damage is unicompartmental and a corresponding axial deviation is found. Particularly the younger patient can benefit from this treatment option in that joint replacement may possibly wholly be avoided or at least postponed to a later stage of life.

Patients diagnosed with hemophilia before the era of home infusion frequently suffer from severe hemarthropathies. Treatment usually relies on physiotherapy for Muscular atrophy and to improve, or at least Maintain, the limited range of Motion of the afflicted joints. For acute pain non-steroidal anti rheumatical drugs (NSAR) are typically given, though in cases of Marked arthritis and hepatobiliary contraindications, efficacy is questionable and the long-term value of this therapy can be disputed. This seems reason enough to contemplate alternative strategies for paintreatment of hemophilic arthropathies. In this context local therapy with Rhenium 186 May be a feasible option.

Transmission of enveloped viruses by blood products prepared from pooled plasma has fortunately become an extremely rare event due to the efficacy of the applied virus inactivation procedures. The inactivation procedures are, however, Much less effective against non-enveloped viruses such as human parvovirus B19 (B19) [for review, 1; 2]. Consequently, B19 infections in association with administration of plasma-derived products are still reported until now [3,4].

The ability to modulate transgene expression in vivo not only will mimic the expression of endogenous genes but is also important for therapeutic and safety reasons in gene therapy protocols. Regulated gene expression systems should be able to induce long-term expression and allow for exogenous control of expression. For this aim, the high gene transfer capacity of adenoviral vectors (Ad) and the high inducibility of the tetracycline-regulatable systems were combined in this study to develop a tetracycline-regulatable Ad vector for expression of human factor IX (hFIX). In vitro studies showed a dose dependent and high induction potential of the hFIX expression system resulting in 65-fold increase in hFIX expression after induction with doxycycline. The use of a liver-specific promoter has been shown to induce a relatively high expression of hFIX compared to CMV promoter in HepG2. In contrast to this, the liver specific promoter showed no hFIX expression when tested in three other non-hepatic human cell lines. The expression of hFIX can either be maintained by continuous induction of doxycycline or up- and down-regulated by mutual presence and absence of doxycycline. When mice were injected with Ad vector for hFIX and induced by doxycycline they showed a therapeutic level of circulating hFIX for one week. These results report on the development of a regulated Ad vectors for the expression of hFIX and demonstrate that FIX gene expression is regulatable in vitro and in vivo. These vectors should be useful not only for gene therapy protocols in haemophilia B but also for diverse applications in gene therapy studies.

For elimination of inhibitors against factor VIII immune tolerance therapy (Bonnor Malmo-protocol) is Mainly used. It requires intravenous injections twice daily up to several Months — while in early infancy antibodies Mostly appear within the first 36 days of treatment [6]. Therefore, in patients with inhibitors or on prophylactic treatment, Port insertion is increasingly recommended (Fig. 1). We describe a particularly complicated case.

Complementary to the CCR5-Delta32 mutation polymorphisms in the genes of CCR2b (CCR2b-V64 I) and stromal derived factor (SDF)-1 (SDF-1 3'A) affect the course of the human immunodeficiency virus (HIV) infection. While the CCR5-Delta32 mutation is also increased in chronic hepatitis C virus (HCV) infection it is unclear, whether the CCR2b-V64 I and the SDF-1 3'A polymorphisms also are associated with chronic HCV infection.We analyzed the frequencies of the CCR2b-V64I and SDF1 - 3'A mutation in patients with HIV/HCV coinfection (n = 130), HIV infection (n = 105), HCV infection (n = 153) and 112 healthy blood donors. We stratified each group into homozygous mutations, heterozygous mutations and homozygous wild types, respectively. The resulting subsets were compared with respect to HIV and HCV loads, CD4 and CD8 cell counts.The mutant SDF1 - 3'A allele was found at 20.3 % frequency in patients with HCV infection and at 20.4 % frequency in patients with HIV/HCV coinfection, respectively. It was present in 27.1 % of the patients with HIV infection and 27.9 % of the healthy controls (not significant). The number of SDF-1 3) A homozygous patients was highest in patients with HIV/HCV coinfection and significantly different compared to the Hardy-Weinberg equilibrium (p = 0.010, chi (2) = 9.15). However, CD4- and CD8-cell counts or viral loads were not affected by this mutation. The frequency of the CCR2b-V64 I allele was similar in all patient groups. However, CCR2b-V64 I heterozygous patients showed HIV loads that were threefold lower than in CCR2b wildtype patients (22.9 x 103 vs. 6.4 x 103 copies/ml, not significant). Furthermore, hepatitis C viral loads were reduced roughly by 30 %.These results suggest that the SDF1 - 3'A and CCR2b-V64I mutations do not affect the course of HCV and HIV/HCV infection in the same manner as does the CCR5-Delta32 mutation.

The severe clotting defects associated with the diagnosis of severe haemophilia A and B require a quality management and quality assurance system designed to avoid both bleeding sequelae (such as damaged joints) through early on-demand or prophylactic treatment in a home-care setting, and side-effects such as infectious diseases (hepatitis A-G and human immunodeficiency virus), allergic reactions, haemolysis and if possible inhibitor formation, by using highly purified, virus-inactivated or recombinant products in which the factor VIII and IX proteins are as natural as possible. As the intravenous injection of the required clotting factor is entrusted to the patients in home treatment, the haemophilia centre has to check treatment protocols and, when necessary, joint and muscle status. In addition, it is imperative to ensure the safety of the product, and checks must be carried out to make sure that batch numbers are recalled as soon as possible if side-effects are observed. These are the reasons for several Acts of Parliament in Germany requiring special treatments and regular checks (the Disabled Act, recommendations by the German Medical Council, the Transfusion Act). Thus, at the haemophilia centre in Bonn we have established a special quality management and quality assurance system taking into account the great number of patients (800), the often considerable distance between the centre and the patient, and the aforementioned regulations and laws. Quality management involves dealing with daily practicalities such as 24-h availability of a physician, medical technologist and nurse, careful instruction of patient and family in home care, genetic counselling, regular laboratory tests (especially recovery time, half-life, inhibitors and gene defects, clinical chemistry and serology) and clinical investigations (especially joint and muscle status). It also includes co-operation with family doctors and different departments at our university hospital (e.g. orthopaedic, microbiology), daily conferences with staff, information for nursery schools, schools, training institutions and/or the workplace in case of emergency, and cooperation with German haemophilia foundations. For quality assurance, several self-controlling systems are in place, such as distribution of concentrate, laboratory data, treatment protocols, joint and muscle status and bleeding tendencies. All these and more are double-checked and interactive, controlling data and activities with the help of EDP. Exceptional staff motivation and patient compliance are important for this quality system.

In view of an increasing tendency in prosthetic management of haemophilic arthropathy, we intended to evaluate the efficacy of corrective osteotomy of the hip, specifically taking long-term clinical and radiographic results into consideration. Eleven hips affected by haemarthropathy in nine patients suffering from severe haemophilia A were treated with an intertrochanteric varus osteotomy. The average follow-up period was 15 small middle dot4 years. The preoperative clinical score of the Advisory Committee of the World Federation of Haemophilia was 5 small middle dot3 points (range 4-7) and the Pettersson score was 6 small middle dot4 points (range 2-10). The average WFH score at follow-up had increased to 3 small middle dot6 points. Seven hips showed clinical improvement, two hips showed a postoperative deterioration while a further two hips remained unchanged. The Petterson score increased to an average of 7 small middle dot7 points. Here the radiographs of six patients indicated postoperative deterioration, three remained unaltered and two showed improvement. Our study cannot conclusively answer whether intertrochanteric varus osteotomy for haemophilic arthropathy of the hip is always a feasible alternative to joint arthroplasty. The decision for or against this procedure must be individually assessed and the patient must be thoroughly informed about the advantages and disadvantages of both procedures.