Structure functional insights into calcium binding during the activation of coagulation factor XIII A.

The dimeric FXIII-A2, a pro-transglutaminase is the catalytic part of the heterotetrameric coagulation FXIII-A2B2 complex that upon activation by calcium binding/thrombin cleavage covalently cross-links preformed fibrin clots protecting them from premature fibrinolysis. Our study characterizes the recently disclosed three calcium binding sites of FXIII-A concerning evolution, mutual crosstalk, thermodynamic activation profile, substrate binding, and interaction with other similarly charged ions. We demonstrate unique structural aspects within FXIII-A calcium binding sites that give rise to functional differences making FXIII unique from other transglutaminases. The first calcium binding site showed an antagonistic relationship towards the other two. The thermodynamic profile of calcium/thrombin-induced FXIII-A activation explains the role of bulk solvent in transitioning its zymogenic dimeric form to an activated monomeric form. We also explain the indirect effect of solvent ion concentration on FXIII-A activation. Our study suggests FXIII-A calcium binding sites could be putative pharmacologically targetable regions.

Identification of Potential Novel Interacting Partners for Coagulation Factor XIII B (FXIII-B) Subunit, a Protein Associated with a Rare Bleeding Disorder.

Coagulation factor XIII (FXIII) is a plasma-circulating heterotetrameric pro-transglutaminase complex that is composed of two catalytic FXIII-A and two protective/regulatory FXIII-B subunits. FXIII acts by forming covalent cross-links within a preformed fibrin clots to prevent its premature fibrinolysis. The FXIII-A subunit is known to have pleiotropic roles outside coagulation, but the FXIII-B subunit is a relatively unexplored entity, both structurally as well as functionally. Its discovered roles so far are limited to that of the carrier/regulatory protein of its partner FXIII-A subunit. In the present study, we have explored the co-presence of protein excipients in commercial FXIII plasma concentrate FibrogamminP by combination of protein purification and mass spectrometry-based verification. Complement factor H was one of the co-excipients observed in this analysis. This was followed by performing pull down assays from plasma in order to detect the putative novel interacting partners for the FXIII-B subunit. Complement system proteins, like complement C3 and complement C1q, were amongst the proteins that were pulled down. The only protein that was observed in both experimental set ups was alpha-2-macroglobulin, which might therefore be a putative interacting partner of the FXIII/FXIII-B subunit. Future functional investigations will be needed to understand the physiological significance of this association.

Recombinant factor VIII products and inhibitor development in previously untreated patients with severe haemophilia A: Combined analysis of three studies.

INTRODUCTION: Standard treatment of congenital haemophilia A is based on replacement therapy with coagulation factor VIII (FVIII) products. A major complication of FVIII therapy is the occurrence of IgG alloantibodies (inhibitors) that neutralize FVIII activity. AIM: The aim of the analysis was estimating the risk of high-titre inhibitor associated with the second-generation full-length product compared to third-generation full-length product and other recombinant FVIII (rFVIII). METHODS: We conducted a combined analysis of individual patient data from three large studies in previously untreated patients (PUPs) with severe haemophilia A. RESULTS: A total of 1109 PUPs were treated from 1993 to 2013 including 787 PUPs treated from 2004 onwards (primary analysis cohort). A total of 322 patients (29.0%) developed an inhibitor, of which 192 (17.3%) a high-titre inhibitor. In the primary analysis set, 29.9% of patients developed an inhibitor and 17.2% a high-titre inhibitor. The combined analysis indicated a lower risk of high-titre inhibitor development for the third-generation rFVIII product compared to the second-generation rFVIII product (primary analysis: adjusted hazard ratio (HR) = 0.72, 95% CI: 0.49 to 1.06). Adjusted HR for all inhibitor development was significantly lower for the third-generation product compared to the second-generation product. CONCLUSION: The trend of an increased risk of inhibitor development in PUPs for one recombinant product illustrates that extrapolation from one recombinant factor VIII product to other products might not be justified.

Free factor XIII activation peptide (fAP-FXIII) is a regulator of factor XIII activity via factor XIII-B.

In a factor XIIIa (FXIIIa) generation assay with recombinant FXIII-A2 (rFXIII-A2 ) free FXIII activation peptide (fAP-FXII) prolonged the time to peak (TTP) but did not affect the area under the curve (AUC) or concentration at peak (CP). Addition of recombinant factorXIII-B2 (rFXIII-B2 ) restored the characteristics of the FXIIIa generation parameters (AUC, TTP and CP) to those observed for plasma FXIII (FXIII-A2 B2 ). FXIII-A2 B2 reconstituted from rFXIII-A2 and rFXIII-B2 showed a similar effect on AUC, TTP and CP in the presence of fAP-FXII as observed for plasma FXIII-A2 B2 , indicating a role for FXIII-B in this observation. An effect of fAP-FXIII on thrombin, the proteolytic activator of FXIII, was excluded by thrombin generation assays and clotting experiments. In a purified system, fAP-FXIII did not interfere with the FXIIIa activity development of thrombin-cleaved rFXIII-A2 (rFXIII-A2 ') also excluding direct inhibition of FXIIIa. However, FXIIIa activity development of FXIII-A2 'B2 was inhibited in a concentration-dependent manner by fAP-FXIII, indicating that an interaction between AP-FXIII and FXIII-B2 contributes to the overall stability of FXIII-A2 'B2 . In addition to its well-known role, FXIII-B also contributes to FXIII-A2 B2 stability or dissociation depending on fAP-FXIII and calcium concentrations.

Evaluation of Risk Minimisation Measures for Blood Components - Based on Reporting Rates of Transfusion-Transmitted Reactions (1997-2013).

BACKGROUND: To assess the impact of safety measures, we compared reporting rates of transfusion-related reactions before and after the implementation of six measures in 1999, 2004, 2006, 2008 and 2009. METHODS: Reporting rates of transfusion-transmitted bacterial infection (TTBI), viral infection (TTVI) and immune-mediated transfusion-related acute lung injury (TRALI) were calculated on the basis of confirmed annual reports and distributed blood components. RESULTS: The introduction of HCV NAT testing caused a significant reduction of HCV reporting rate from 1:0.6 to 1:83.16 million administered blood components (p < 0.0001), donor screening for antibodies to hepatitis B core antigen caused a reduction of HBV reporting rate from 1:2.90 to 1:10.70 million units (p = 0.0168). A significant reduction from 1:0.094 to 1:2.42 million fresh frozen plasma (FFP) units could also be achieved by risk minimisation TRALI measures (p < 0.0001). Implementation of pre-donation sampling did not result in a significant decrease in TTBI, whereas limitation of shelf life for platelet concentrate (PC) minimised the TTBI reporting rate from 1:0.088 to 1:0.19 million PC units (p = 0.041). For HIV NAT pool testing, no significant reduction in HIV transmission was found due to very low reporting rates (1:10 million versus 1:27 million blood components, p = 0.422). CONCLUSION: On the basis of haemovigilance data, a significant benefit could be demonstrated for four of six implemented safety measures.

Risk of Guillain-Barré syndrome following pandemic influenza A(H1N1) 2009 vaccination in Germany.

PURPOSE: A prospective, epidemiologic study was conducted to assess whether the 2009 pandemic influenza A(H1N1) vaccination in Germany almost exclusively using an AS03-adjuvanted vaccine (Pandemrix) impacts the risk of Guillain-Barré syndrome (GBS) and its variant Fisher syndrome (FS). METHODS: Potential cases of GBS/FS were reported by 351 participating hospitals throughout Germany. The self-controlled case series methodology was applied to all GBS/FS cases fulfilling the Brighton Collaboration (BC) case definition (levels 1-3 of diagnostic certainty) with symptom onset between 1 November 2009 and 30 September 2010 reported until end of December 2010. RESULTS: Out of 676 GBS/FS reports, in 30 cases, GBS/FS (BC levels 1-3) occurred within 150 days following influenza A(H1N1) vaccination. The relative incidence of GBS/FS within the primary risk period (days 5-42 post-vaccination) compared with the control period (days 43-150 post-vaccination) was 4.65 (95%CI [2.17, 9.98]). Similar results were found when stratifying for infections within 3 weeks prior to onset of GBS/FS and when excluding cases with additional seasonal influenza vaccination. The overall result of temporally adjusted analyses supported the primary finding of an increased relative incidence of GBS/FS following influenza A(H1N1) vaccination. CONCLUSIONS: The results indicate an increased risk of GBS/FS in temporal association with pandemic influenza A(H1N1) vaccination in Germany.

Factor XIIIa generation assay: a tool for studying factor XIII function in plasma.

Triggering the extrinsic coagulation pathway in plasma and using a fluorogenic factor XIIIa (FXIIIa) substrate for continuously monitoring FXIIIa activity, an FXIIIa generation curve is obtained. The parameters area under the curve (AUC), time to peak (TTP), and concentration at peak (CP) were calculated. In dilutions of normal plasma in FXIII-deficient plasma, AUC and CP showed linear dose-response relationships, whereas TTP increased from 9.9 min for 25% FXIII to 11.6 min for 100% FXIII. Three FXIII-A preparations (rFXIII, rFXIII(V34L), and cellular FXIII [cFXIII]) showed a linear dose response for AUC and CP. The TTP increased slightly for rFXIII from 13.5 to 15.0 min, but surprisingly for cFXIII TTP increased concentration dependently from 13.5 to 28.7 min. Adding 5 µg/ml FXIII-B at a concentration of 1U of FXIII-A increased the AUC for rFXIII(V34L) and cFXIII by approximately 20% and accelerated TTP from 27.3 to 20.8 min for cFVIII, indicating a supportive function of FXIII-B in orientating cFXIII-A for thrombin cleavage. A commercial assay quantifying FXIII after complete activation in a restricted time window did not reveal differences in the cFXIII preparation with or without FXIII-B. The FXIIIa generation assay provides additional information about activation and function of FXIII. This advantage was underlined in experiments with an irreversible FXIIIa inhibitor.

Effect of Safety Measures on Bacterial Contamination Rates of Blood Components in Germany.

SUMMARY: Requirements for bacterial testing of blood components on a defined quantity as part of routine quality control were introduced in Germany by the National Advisory Committee Blood of the German Federal Ministry of Health in 1997. The philosophy was to establish standardized methods for bacterial testing. Numerous measures to reduce the risk of bacterial contamination were implemented into the blood donation and manufacturing processes between 1999 and 2002. German Blood establishments performed culture-based bacterial testing on random samples of platelet concentrates (PCs), red blood cells (RBCs) and fresh frozen plasma (FFP) and reported data out of the production periods 1998, 2001 and 2005/2006. While the bacterial contamination rate of apheresis PCs remained nearly unchanged, it decreased by 70% for pooled PCs to a rate of 0.158% in the last observation period. Leukocyte-depleted RBCs with diversion of the initial blood volume showed a contamination rate of 0.029% which is significantly lower than that of RBCs without leukocyte depletion and diversion (0.157%). The contamination rate of plasma decreased by 80%. Preventive measures resulted in a significant reduction of bacterial contamination of blood components. Long-term monitoring with standardized methods for bacteria testing supports evaluation of the cumulative effect of contamination reducing measures.

Evaluation of Quality Parameters for Cord Blood Donations.

SUMMARY: BACKGROUND: Umbilical cord blood (CB) is widely used for hematopoietic stem cell transplantation and holds promise for the development of innovative medicinal products. In order to find out whether the conditions for collection and storage before processing might have an impact on the quality of CB preparations, viability and the clonogenic potential were assessed. METHODS: CB was collected under field conditions. Flow cytometry was used to determine leukocytes, CD34/CD45+ cells, viability, and nucleated red blood cells (NRBC). Clonogenic activity was determined using isolated mononuclear cells (MNC). RESULTS: Neither plasma citrate concentrations nor storage temperature (within 24 h) affected cell viability or colony formation. After storage for 49-80 h, leukocyte viability declined by about 16% compared to CB stored up to 24 h. In contrast, the clonogenic activity and CD34/CD45+ cell content were not affected. A higher gestational age was associated with a lower yield of clonogenic activity compared to midterm deliveries. NRBC varied widely (median 7.3%; range 0.63-17.3%) without relation to gestational age or colony formation. There was a close correlation between the percentage of viable CD34/CD45+ cells and colony formation (r = 0.77 for CFU-GM; r = 0.75 for CFU-C). CONCLUSIONS: The content of viable CD34/CD45+ cells represents the clonogenic activity of CB preparations. Therefore, determination of viable CD34/CD45+ cells should be generally performed as a routine quality control assay.

Impact of vCJD on blood supply.

Variant Creutzfeldt-Jakob disease (vCJD) is an at present inevitably lethal neurodegenerative disease which can only be diagnosed definitely post mortem. The majority of the approximately 200 victims to date have resided in the UK where most contaminated beef materials entered the food chain. Three cases in the UK demonstrated that vCJD can be transmitted by blood transfusion. Since BSE and vCJD have spread to several countries outside the UK, it appears advisable that specific risk assessments be carried out in different countries and geographic areas. This review explains the approach adopted by Germany in assessing the risk and considering precautionary measures. A fundamental premise is that the feeding chain of cattle and the food chain have been successfully and permanently cleared from contaminated material. This raises the question of whether transmissions via blood transfusions could have the potential to perpetuate vCJD in mankind. A model calculation based on actual population data showed, however, that this would not be the case. Moreover, an exclusion of transfusion recipients from blood donation would add very little to the safety of blood transfusions, but would have a considerable impact on blood supply. Therefore, an exclusion of transfusion recipients was not recommended in Germany.

Neopterin levels during the early phase of human immunodeficiency virus, hepatitis C virus, or hepatitis B virus infection.

BACKGROUND: A study was conducted to assess the diagnostic sensitivity of neopterin screening of blood donors with regard to the detection of window-phase specimens of human immunodeficiency virus (HIV), hepatitis C virus (HCV), and hepatitis B virus (HBV) infection. STUDY DESIGN AND METHODS: In total, 1002 diagnostic window-phase specimens from 98 seroconversion panels (29 HIV-1, 52 HCV, and 17 HBV) were analyzed with viral antigen detection, viral nucleic acid amplification testing (NAT), and neopterin quantitation assays. The study was completed by the analysis of 92 anti-hepatitis B core antigen (HBc)-reactive and 103 alanine aminotransferase (ALT)-elevated blood donor specimens. RESULTS: A significant association between elevated neopterin concentrations and the very early phase of HIV-1 infection was found. No significant correlation could be observed between neopterin levels and the early phase of HCV or HBV infection. Neopterin concentration was not increased in specimens from blood donors with anti-HBc reactivity or ALT elevation. CONCLUSIONS: Neopterin screening of blood donors may identify window-phase cases of HIV, but not of HCV or HBV infection. The diagnostic sensitivity of neopterin screening during the HIV window phase is similar to that of the p24 antigen test. With the introduction of viral NATs in blood screening, there is no additional benefit of neopterin screening with regard to the three blood-borne viruses HIV, HCV, and HBV. Acute phases of other infectious agents, however, have been reported to be detected by neopterin enzyme-linked immunosorbent assays.

An introductory note to CHMP guidelines: choice of the non-inferiority margin and data monitoring committees.

The Committee on Medical Products for Human Use (CHMP) has recently issued two new guidance documents that are of particular note to statisticians. The purpose of this short note is to give a little background to the origins of these documents and a flavour of some of their most important features. The two guidelines are reproduced as the next two papers in the journal.

[Serological test methods as replacement for infection trials in piglets to test the potency of E. coli vaccines for sows (dams)]. / Serologische Testmethoden als Ersatz fuer Infektionsversuche an Ferkeln zur Wirksamkeitspruefung von E.coli-Muttertierimpfstoffen.

Escherichia coli (E. coli) related diarrhoea poses a major problem in piglet production. Vaccination of dams increases the antibody titre in their milk against relevant adhesion antigens. Transfer of maternal antibodies via the milk can serve to protect the suckling piglets from the disease. However, a marketing authorization for veterinary use of these vaccines requires potency determination as assayed in infection trials in piglets. Enzyme immunoassays testing the antibodies in blood serum and colostrum samples from vaccinated dams against the relevant adhesion antigens, F4ab, F4ac, F6 and F41, were developed to replace the very distressing infection experiments by in vitro methods. Eight vaccines authorized in Germany were included in the trials. Vaccinated sows showed significantly higher antibody titres in blood and colostrum than unvaccinated control animals. Thus the enzyme immunoassays appear to be suitable to detect antibodies against four important adhesion antigens, and can be used to replace the distressing infection trials in piglets.

Quality assurance of C. perfringens epsilon toxoid vaccines--ELISA versus mouse neutralisation test.

Clostridium (C.) perfringens is a Gram-positive anaerobic spore-forming bacterium. Disease caused by C. perfringens infection is called enterotoxaemia. C. perfringens strains are classified on the basis of the lethal exotoxins formed by the bacteria. Epsilon toxin is one of the major lethal toxins and is formed by C. perfringens types B and D. C. perfringens is an ubiquitous bacterium. Infection occurs via food, water, animal litter or soil. Affected animals include mainly sheep, pigs and cattle. C. perfringens infection manifests as pulpy kidney disease and diarrhoea in suckling lambs. Enterotoxaemia development is peracute in most cases. Animals die suddenly while grazing on the pasture, without any prior signs of disease. Therefore, treatment is possible only in very rare cases. Suitable immunoprophylactic measures are the treatment of choice to combat the disease: Vaccines and immunosera have therefore been used extensively for a long time. The requirements for quality, efficacy and safety testing of the inactivated vaccines are laid down in the Ph. Eur. in the monograph: Clostridium perfringens vaccines for veterinary use. After a marketing authorisation is attained, the product batches must be tested in laboratory animal models for their potency against all vaccine components (Pharmeuropa, 1997). For potency testing (batch control) of C. perfringens types B and D, the induction of specific antibodies against epsilon toxin in rabbits must be verified. For this purpose, 10 rabbits are immunised twice with the product to be tested. Their blood is taken 14 days after the last immunisation and the serum is pooled. The pooled serum is then tested for its protective effect. This is done by means of the toxin neutralisation test in mice (optionally also in guinea pigs) in comparison with an international reference serum. The evaluation criterion is the death rate of the mice in the test and reference groups after administration of lethal doses of epsilon toxin. The exact efficacy of the test serum is given in International Units (IU). The tested serum must show a minimum content of 5 IU. This in vivo method requires a very high number of experimental animals. Approximately 400 mice (or 50 guinea pigs) are used per vaccine batch. The monograph for C. perfringens vaccines, which has recently been revised, expressly indicates that a validated serological method may be used for batch testing. In addition, a reference serum known as clostridium multicomponent serum has been available since 2000. The objective is to test vaccine batches against this reference and by means of a competitive ELISA developed in the precursor project, using a monoclonal antibody for direct determination of specific antitoxins in rabbit sera. This ELISA method was subjected to an international validation to verify whether the protocol and the precision can be transferred within and between the participating laboratories.

Suitability of temperature-sensitive transponders to measure body temperature during animal experiments required for regulatory tests.

Body temperature is a clinical parameter in vaccine quality control to detect systemic side-effects or to monitor progression of infectious diseases. Moreover, changes in body temperature are used as clinical parameters to define humane endpoints in animal experiments. However, measuring body temperature via the rectal route can be troublesome and distressing to the animal. Non-invasive measurement methods were developed in recent years. The aim of this investigation was to study and to compare rectally measured body temperature with data obtained with implanted temperature-sensitive transponders (TST) in mice, guinea pigs, rabbits and pigs under the controlled conditions of regulatory testing.