Clottable fibrinogen needs to be detected and quantified e.g. when producing fibrinogen based products, such as fibrinogen concentrate and fibrin sealants.
The presently disclosed subject matter particularly refers to the preparation of a sample for the detection of clottable fibrinogen by so-called gravimetric assay process according to the European Pharmacopeia 7.0 (also see: Gaffney P. J. and Wong M. Y. Collaborative study of a proposed International Standard for plasma Fibrinogen measurement. Thrombosis and Heamostasis, 68 (4), pp. 428-432, 1992), the process generally including the following steps:                a) adding thrombin solution and EDTA to the tested fibrinogen containing sample, thereby forming a fibrin clot. EDTA (a Ca++ chelator agent) inhibits the activation of FXIII by thrombin to FXIIIa (plasma-transglutaminase), thus preventing the formation of γ-glutamyl-ε-lysine bridge of non-clottable protein to fibrin;        b) drying the formed clot (e.g. by absorption of the liquid on a Whatman paper) followed by several washes e.g. with a saline solution whereby “non-clottable” proteins are removed; and        c) having the remained fibrin clot following step (b) degraded (e.g. by sulfuric acidic, or a mixture of 6M urea and 0.2M NaOH), thereby producing a sample for further use in detection/quantification of fibrinogen therein by any known method e.g. a spectrophotometer measurement.        
Dempfle et. al. (“A 96-well microfiltration assay for measurement of Total Clottable Fibrinogen”; Thromb Haemost, 1999, 81, 264-267) discloses a gravimetric assay process generally including the following steps:                a) adding thrombin solution to a fibrinogen containing sample disposed on a filtration membrane, in order to form a fibrin clot on that membrane;        b) washing the clot formed on the filtration membrane in step (a) and drying it by applying vacuum to the membrane at its side opposite to that holding the fibrin clot, whereby “non-clottable” proteins are removed through the filtration membrane, leaving thereon dried clottable fibrin; and        c) dissolving the fibrin clot remained in step (b) in solvent solution, producing thereby the sample for its further use in detection/quantification of fibrinogen therein by any known method.        
In Dempfle et. al. a 96-well microfiltration assay is used, wherein each well comprises a 1.2 μm pore size hydrophilic membrane, preconditioned with a detergent. In this process, steps (a) and (b) above are performed within the array, whilst after step (b) the membranes with fibrin clot adhered thereto are removed from the assay, and the solubilization of the fibrin clot from each of the 96 membranes (step (c) above) is performed in a separate test tube, one membrane with adherent fibrin clot per such tube.
USPTO Backgrounds
USPTO Backgrounds