Thrombin Generation Capacity of Methylene Blue-Treated Plasma Prepared by the Theraflex MB Plasma System.

BACKGROUND: Methylene blue (MB) / light treatment is a well-known procedure for the inactivation of pathogens in fresh frozen plasma (FFP). Aim of the current study was to investigate the thrombin generation (TG) characteristics and quality of MB plasma prepared by the Theraflex MB Plasma System. METHODS: Single donor plasma units (n = 18) were MB/light-treated, with sampling before and after processing. Preparation included leukocyte depletion, addition of MB pill prior to illumination, and depletion of MB and photoproducts by filtration. Different plasma parameters and TG were measured. TG additionally was determined in solvent/detergent plasma (n = 8). RESULTS: MB/light treatment significantly affected factors V, VIII and XI, which were decreased by 9-18%. While the antigen level was not affected, fibrinogen according to Clauss was decreased by 7%, correlating with a 12% prolongation of TT and RT. The total amount of free thrombin generated, given as 'area under the curve' (AUC), was comparable for untreated (93 +/- 18% of normal plasma) and MB/light-treated plasma (95 +/- 20%). Also peak thrombin concentration was not significantly affected by treatment (94 +/- 11% (untreated) vs. 96 +/- 12% (treated)). The 'time to peak' value (TTP) was 105% of normal plasma for untreated FFP and 89% for MB-treated plasma. CONCLUSION: For plasma treated with the Theraflex MB Plasma System no profound influence of MB/ light treatment on the characteristics of thrombin generation was detected. In concordance with data from the literature, coagulation factors V, VIII and XI were decreased due to MB/ light treatment. Decrease was less than 20%.

A novel assay for factor XIII based on cross-linking of synthetic peptides: analysis of different substrates.

A novel assay for factor XIII is described that utilizes exclusively small synthetic peptides as substrates for the cross-linking reaction catalyzed by activated factor XIII (FXIIIa). The acyl donor substrate (selection peptide) is immobilized on a microplate via biotin while the acyl acceptor substrate (detection peptide) is labeled with the fluorochrome Oregon green to allow sensitive detection without the need for secondary enzyme systems for signal amplification. Starting with an amino acid sequence from the fibrin gamma-chain (GQQHHLGGAKQAGDV) as a prototype peptide, the influence of amino acid exchanges were investigated with respect to their impact on the FXIIIa-catalyzed reaction. It was found that FXIIIa readily accepts a broad range of substrate peptides, with a proline neighboring the essential lysine having the most detrimental effect. The assay appears to be valuable for the molecular characterization of factor XIII and may be used for a deeper investigation into the substrate requirements of this final enzyme of wound repair, and eventually also for the characterization of other transglutaminases.

Factor VIII determination in patient's plasma and concentrates: a novel test equally suited for both matrices.

A novel assay for the determination of factor VIII (FVIII) is described. The assay uses a fluorescence-based detection system comparable with the common chromogenic test. At the same time, the assay is analogous to the clotting test as it does not involve pre-activation of FVIII. The assay was adjusted to perform equally well with patient's plasma and FVIII concentrates as samples. The combined employment of two different FVIII-deficient plasmas turned out to be of crucial importance in order to render the matrices as similar as possible to patient's plasma and, simultaneously, to obtain maximum sensitivity. Samples with a FVIII content down to 0.01 IU/ml are readily measured as are samples with a FVIII content of 1 IU/ml or somewhere in between. Upon dilution of samples, concentrates and plasma exhibited the same dose-response characteristics.

[An in vitro alternative to the mouse neutralisation assay for potency testing of betatoxoid containing Clostridium, perfringens type B and type C vaccines for veterinary use]

The quality control of Clostridium (C.) perfringens type B and type C vaccines requires animal experiments according to European Pharmacopoiea monograph 363. For potency estimation, the vaccine is first administered to rabbits. In a second step antibodies from these rabbits against C. perfringens betatoxin are measured quantitatively in a mouse neutralisation assay using lethal doses of betatoxin for the challenge. We report about the development of an in vitro assay enabling specific and reproducible measurement of antibodies against C. perfringens betatoxin in rabbit sera. A Capure-Enzyme Linked Immuno Sorbent Assay (ELISA) using a monoclonal antibody against betatoxin as catching antibody was used. A rabbit serumpool freeze dried in 3500 aliquots was always used as reference. This reference serum can be supplied for further national or international collaborative studies. The estimation of relative potency of unknown sera in a parallel line assay was calculated with a computer programme provided by the World health organisation (WHO). The capture-ELISA did not show unspecific reactivity with pre-vaccination sera of cross-reactivity with sera from rabbits immunised with other clostridial antigens e.g. C. perfringens type D, C. chavoei or C. tetani. Reproducibility studies focused on the linear parts of the dose-response curves resulted in intra-assay coefficient of variations of less then 10%. The inter-assay coefficient varied between 12-25% depending on the serum dilutions used. Correlation studies between the result of the animal experiment (only one test) and the capture-ELISA (10 repetitions) from four rabbit serum pools revealed a coefficient of correlation of 0.81-0.84 depending on the basis for calculation of r (Mean or Median from ELISA repetitions). Therefore this test may be a suitable alternative for the currently required mouse neutralisation assay. For acceptance of this test by the European Pharmacopoiea further validation studies are necessary.

[Detection of antibodies against Pasteurella multocida toxin with a cell culture method and an enzyme immunoassay]

Pasteurella multocida toxoid is the most important antigen in vaccines against progressive atrophic rhinitis in pigs. Testing antibodies against Pasteurella multocida toxin in a cell culture neutralisation assay on embryonic bovine lung cells and a modified, commercially available enzyme-linked immunosorbent assay (DAKO, Denmark) is sensitive and gives good reproducible results. Determination of antitoxin antibodies in swine and guinea pigs simultaneously in both methods resulted in good coefficients of correlation (r = 0.88 and 0.93). Induction of antibodies to Pasteurella multocida toxin by thirty batches of ten toxoid containing vaccines was tested by subcutaneous application of one fifth pigdose (0.4-1 ml) twice in intervals of three weeks. The animals showed neither signs if illness nor significant local or systemic reactions. Three weeks after the second immunisation 25 batches induced titres being at least 2 log2 dilutions higher than a parallel titrated reference serum (mean titre of reference serum was 1:23.26