Wolfgang Muntean

Factor VIII Products and Inhibitor Development in Severe Hemophilia A

BACKGROUND For previously untreated children with severe hemophilia A, it is unclear whether the type of factor VIII product administered and switching among products are associated with the development of clinically relevant inhibitory antibodies (inhibitor development). METHODS We evaluated 574 consecutive patients with severe hemophilia A (factor VIII activity, <0.01 IU per milliliter) who were born between 2000 and 2010 and collected data on all clotting-factor administration for up to 75 exposure days. The primary outcome was inhibitor development, which was defined as at least two positive inhibitor tests with decreased in vivo recovery of factor VIII levels. RESULTS Inhibitory antibodies developed in 177 of the 574 children (cumulative incidence, 32.4%); 116 patients had a high-titer inhibitory antibody, defined as a peak titer of at least 5 Bethesda units per milliliter (cumulative incidence, 22.4%). Plasma-derived products conferred a risk of inhibitor development that was similar to the risk with recombinant products (adjusted hazard ratio as compared with recombinant products, 0.96; 95% confidence interval [CI], 0.62 to 1.49). As compared with third-generation full-length recombinant products (derived from the full-length complementary DNA sequence of human factor VIII), second-generation full-length products were associated with an increased risk of inhibitor development (adjusted hazard ratio, 1.60; 95% CI, 1.08 to 2.37). The content of von Willebrand factor in the products and switching among products were not associated with the risk of inhibitor development. CONCLUSIONS Recombinant and plasma-derived factor VIII products conferred similar risks of inhibitor development, and the content of von Willebrand factor in the products and switching among products were not associated with the risk of inhibitor development. Second-generation full-length recombinant products were associated with an increased risk, as compared with third-generation products. (Funded by Bayer Healthcare and Baxter BioScience.).

Low tissue factor pathway inhibitor (TFPI) together with low antithrombin allows sufficient thrombin generation in neonates

Summary.  Neonates have an excellent hemostasis despite, in comparison to adults, markedly decreased and delayed ability to generate thrombin. Only 30–50% of peak adult thrombin activity can be produced in neonatal plasma by means of conventional in vitro assays. We show that in contrast to conventional activation, activation with small amounts of lipidated tissue factor (<10 pmol L−1) results in shorter clotting times and faster activated factor X‐ and thrombin generation in neonates compared with adults due to the concomitant action of low tissue factor pathway inhibitor and antithrombin. The concentrations of both inhibitors in cord plasma are approximately 50% of the respective adult values. After addition of 2.5 pmol L−1 lipidated tissue factor, cord plasma clotted ∼90 s earlier than adult plasma and the amount of free thrombin generated was ∼90% of adult value (291 ± 14 vs. 329 ± 16 nmol L−1 min−1, P < 0.01). Our results might help to explain the clinically observed excellent hemostasis of neonates despite low levels of procoagulant factors.

Activation of the clotting system during extracorporeal membrane oxygenation in term newborn infants

OBJECTIVES To determine the degree of clotting activation that occurs with the usual anticoagulation regimen with systemic heparinization. METHODS To allow a standardized comparison of the patients, this study focused on the first 48 hours of extracorporeal membrane oxygenation (ECMO) in term newborn infants. The ECMO perfusion circuit consisted of a roller pump, silicone membrane lungs, and silicone rubber tubing. Coagulation was controlled routinely by measuring prothrombin time, fibrinogen, antithrombin III, and reptilase time. Platelet counts, activated clotting time, and heparin concentration were controlled regularly. The following specific activation markers of the clotting system were measured: prothrombin activation fragment 1 + 2(F1+2), thrombin-antithrombin III complexes, and D-dimer. Measurements were done before the start of ECMO, after 5 minutes, and at hours 1, 2, 3, 4, 6, 12, 24 and 48. RESULTS All seven term infants had excessively high levels of clotting activation markers within the first 2 hours of ECMO: F1+2, 11.6(+/- O.9) nmol/L (mean +/- SEM); thrombin-antithrombin, 920(+/- 2.2) microg/L; D-dimer, 15.522(+/- 3.689) ng/L. During the next 46 hours of ECMO, F1+2 and thrombin-antithrombin III complexes decreased from those high values, whereas D-dimer did not. The increase of activation markers was accompanied by low fibrinogen, low platelet counts. and prolongation of reptilase time. CONCLUSIONS These findings fit the pattern of consumptive coagulopathy during neonatal ECMO, especially in the first 24 hours.

Calibrated automated thrombin generation in normal uncomplicated pregnancy

Pregnancy is associated with substantial changes in the haemostatic system and a six-fold higher incidence of venous thromboembolism. Conventional global tests, such as prothrombin time and activated partial thromboplastin time, do not definitely detect this hypercoagulable condition. We investigated whether the changes in haemostatic system during pregnancy are reflected in the calibrated automated thrombography (CAT). Thrombin generation was measured in platelet-poor plasma (PPP) of 150 healthy pregnant women without any pregnancy associated diseases by means of CAT. In addition, prothrombin (FII), antithrombin (AT), protein S, protein C, tissue factor pathway inhibitor (TFPI), plasminogen activator inhibitor-1 (PAI-1), thrombin-antithrombin complex (TAT), and prothrombin fragments 1+2 (F1+2) were measured. Endogenous thrombin potential (ETP) and peak of thrombin generation increased significantly with gestational weeks, while lag time and time to peak remained unchanged. A significant increase of PAI-1, TFPI, F1+2 and TAT as well as a significant decrease of free protein S, protein S antigen, and protein S activity was observed. Levels of AT and protein C remained stable during pregnancy. Division of population in trimester of pregnancy and analysis of differences between the trimesters showed rather similar results. Our study shows that endogenous thrombin potential does increase with duration of normal uncomplicated pregnancy. Whether parameters of continuous thrombin generation will correlate with thrombembolic disease remains to be shown.

Practical guidelines for the clinical use of plasma

Despite differences in the composition of fresh frozen plasma (FFP) and solvent/detergent-treated plasma, prospective controlled clinical trials have not revealed any significant difference in clinical efficacy and tolerance between the two types of plasma. Evidence of the clinical efficacy of plasma is mainly based on expert opinion, case reports, and on controlled and uncontrolled observational studies. The application of plasma without laboratory analysis to verify the coagulopathy is normally not justified. With the exception of emergency situations when timely clotting assay results are not available, the administration of plasma in coagulopathy must be verified both clinically and by laboratory analysis before plasma is administered. The rapid infusion of at least 10 ml plasma/kg of body weight is required to increase the respective plasma protein levels significantly. Based on the present state of knowledge, plasma is indicated for complex coagulopathy associated with manifest or imminent bleeding in massive transfusion, disseminated intravascular coagulation, and liver disease. Therapeutic plasma exchange with 40 ml plasma/kg of body weight is the treatment of first choice in acute thrombotic-thrombocytopenic purpura-adult hemolytic uremic syndrome (TTP-HUS). A rare indication is the treatment or prevention of bleeding in congenital factor V or factor XI deficiency, plasma exchange in neonates with severe hemolysis or hyperbilirubinemia, and filling of the oxygenator in extracorporeal membrane oxygenation (ECMO) in neonates. Prothrombin complex concentrates should be preferred to plasma for the reversal of oral anticoagulation in emergency situations, since controlled studies have shown a minor efficacy of plasma. Side effects resulting from the administration of plasma are rare but have to be considered.

Shorter PFA-100 closure times in neonates than in adults: role of red cells, white cells, platelets and von Willebrand factor

UNLABELLED In neonates, despite poor platelet function in various in vitro tests, closure times (CTs) in PFA-100 measurements are shorter than in adults. Neonates have a higher polymeric von Willebrand factor (vWF). They also have a higher haematocrit and higher white blood cell count than adults. which may interfere with the evaluation of platelet and vWF function by means of the PFA-100 in neonates. To assess the role of different blood constituents on neonatal CTs, red blood cell, platelet and white blood cell counts in cord blood were modified. These modifications did not provide any evidence that the difference in number between adult and neonatal blood cells was responsible for shorter neonatal CTs. In further experiments, platelets and/or vWF were inhibited by means of abciximab and anti-vWF antibody, and mixing experiments with neonatal platelet-rich and platelet-poor plasma were performed. The results showed that short cord blood PFA-100 CTs were caused by a constituent of neonatal platelet-poor plasma, probably the neonatal high multimeric vWF. CONCLUSION This study demonstrates that CTs in neonates are dependent on the same components, platelets and vWF, as in adults, making it likely that the PFA-100 can be used in neonates in the same way as in adults to investigate platelet and vWF function.

Treatment of children with haemophilia in Europe: a survey of 20 centres in 16 countries.

A survey was made of the current status of treatment of haemophilic boys at 20 centres in 16 European countries and includes approximately 1500 of the estimated 6500 haemophiliacs in the participating countries. Many mild haemophiliacs are not seen, or seen infrequently, at haemophilia centres and this requires study. Nine of 18 centres provide continuous prophylaxis to 80–100% of their patients, five centres provide it to 55–80% and the remaining four centres to 15–40% of the boys. The median dose given was 6240 U kg−1 year−1 (range 3120–7800). Four centres administered only recombinant concentrates to children with severe haemophilia A, while seven centres administered recombinant concentrates to 75–90% and the remaining centres to less than 50% of the boys (two centres < 10%). When asked for the choice of concentrate for a newly diagnosed boy with severe haemophilia A, all but one centre preferred recombinant concentrate. Most boys below 6 years received concentrates via a peripheral vein but three centres preferred a central venous line for 80–100% of the boys. Thirteen of 18 centres applied home treatment to 84–100% of the boys and the remaining five centres to 57–77% of the boys.

Tissue plasminogen activator (alteplase) treatment for femoral artery thrombosis after cardiac catheterisation in infants and children.

OBJECTIVE--To determine the efficacy of fibrinolytic therapy with tissue plasminogen activator (alteplase) in infants and children with arterial thrombosis after cardiac catheterisation. DESIGN--Use of alteplase (Actilyse) in a protocol with prospective data collection. Alteplase was administered to infants and children with arterial thrombosis after cardiac catheterisation. A dose of 0.5 mg/kg/h was given continuously via a peripheral vein for the first hour followed by 0.25 mg/kg/h till clot lysis occurred or treatment had to be stopped because of bleeding complications. SETTING--University hospital, intensive care unit. PATIENTS--17 consecutive infants and children with femoral artery thrombosis after cardiac catheterisation between 1 April 1988 and 31 October 1991. MAIN OUTCOME MEASURE--Reopening of the vessel. RESULTS--Complete clot lysis was achieved in 16 of 17 patients within 4-11 hours after the start of treatment. In one patient only partial lysis occurred. After complete lysis rethrombosis developed in one patient 15 hours after the end of treatment. Bleeding complications were seen in nine patients. These were restricted to the arterial puncture site, except for one who showed mild epistaxis. Three patients had to be treated with packed erythrocytes. CONCLUSIONS--Alteplase was an effective treatment of arterial thrombosis after cardiac catheterisation in infants and children. Further studies are needed to determine whether lower doses will reduce the frequently observed bleeding complications.

Absence of Anti-Human Immunodeficiency Virus Types 1 and 2 Seroconversion after the Treatment of Hemophilia A or von Willebrand's Disease with Pasteurized Factor VIII Concentrate

Patients with hemophilia A or von Willebrands disease who are treated with concentrated preparations of human factor VIII made from unscreened pooled plasma are at substantial risk of contracting human immunodeficiency virus (HIV) infection. The purpose of this study was to investigate whether by treating such patients with a pasteurized factor VIII concentrate that had been heated in aqueous solution at 60 degrees C for 10 hours, HIV infection could be avoided. Eleven hemophilia centers in the Federal Republic of Germany and two in Austria identified 155 eligible patients who had been treated exclusively with pasteurized factor VIII concentrate and had not received any other blood products. Between February 1979 and December 1986 they received a total of 15,916,260 IU of pasteurized factor VIII. The United States was the source of 80 percent of the plasma from which the concentrate was made. By September 1988, these 155 patients had been screened for antibody to HIV type 1 (anti-HIV-1) with a total of 657 tests; all were negative. Sixty-seven patients were also tested once for antibody to HIV type 2 (anti-HIV-2); all these tests were negative as well. It appears that pasteurization effectively inactivates HIV, even in plasma that is likely to be highly contaminated with the virus.

Reassessment of autoreactivity of the broadly neutralizing HIV antibodies 4E10 and 2F5 and retrospective analysis of clinical safety data

Background:The broadly neutralizing recombinant human HIV-1 antibodies 4E10, 2F5 and Igh1b12 are reported to have autoreactive potential, which is significant for HIV-1 vaccine development and passive immunotherapy using these antibodies. Objective:To investigate the clinical relevance of these findings in subjects receiving passive immunotherapy with these antibodies. Methods:Four types of investigations were performed: (1) Investigation of clotting parameters in an ongoing clinical study with 4E10, 2F5 and 2G12. (2) Mixing experiments of pooled plasma with the same antibodies. (3) Retrospective analysis of serum from patients who received passive immunotherapy with 4E10, 2F5 and 2G12 either alone or in combination. (4) Assessment of clinical safety data obtained after 418 infusions with these antibodies. Results:Standard clinical assays confirmed that 4E10 showed low-level cross-reactivity with cardiolipin, while previously reported cardiolipin cross-reactivity for 2F5 could not be confirmed. High serum titers of 4E10 induced mild prolongation of the activated partial thromboplastin time, which resolved with the wash out of 4E10. Neither 2F5 nor 2G12 affected coagulation. Repeated high-dose infusions of the monoclonal antibody combination were well tolerated with no incidence for thrombotic complications after 418 infusions in 39 subjects. Conclusions:Monoclonal antibody 4E10 but not 2F5 or 2G12 showed autoreactive binding specificities. Infusion of 4E10 resulted in transient low anticardiolipin titers. Although an increased thromboembolic risk cannot definitely be excluded, this risk appears to be low and likely depend on underlying disorders.