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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%.

Abstract:The technique of thrombin generation has been established as a promising tool for the diagnosis, risk estimation, and perhaps prognosis of coagulation insufficiencies. Without further experimentation an indication can be obtained if a sample donor carries the risk for haemophilia or thrombophilia, either congenital or acquired. A comparison of two different thrombin generation assays is presented, demonstrating that a currently not commercially available test allows an easier and cheaper calibration than its commercial counterpart. This is achieved by employment of a less efficient but highly specific peptide substrate for thrombin and the involvement of low amounts of analyte (plasma samples).

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.

SummaryA very sensitive and highly reliable test system for the detection of activated coagulation factor IX (FIXa) has been established. This assay system is based on the cleavage of a fluorogenic substrate by activated factor X (FXa) which is generated by FIXa. This assay can be used to process a large number of samples at a time and, being based on the convenient microtiter plate format, can easily be adapted to automated processing for routine screening of large sample numbers.With this assay at hand we determined the FIXa content of different commercially available therapeutic FIX sources, such as high purity FIX (HPFIX) and prothrombin complex concentrates (PCC). Here we demonstrate that PCC from several suppliers do not contain significantly higher levels of FIXa as compared to HPFIX from the same supplier. In fact, there is a tendency for HPFIX to contain more FIXa than PCC. Moreover, HPFIX from certain manufacturers who do not produce PCC are characterized by an exceptionally high content of FIXa. Therefore, the higher thrombogenic potential of PCC which is well documented clinically cannot be explained solely – if at all – by an increased content of FIXa. Rather, it will be necessary to identify other components responsible for this phenomenon.