In vitro methods for screening for blood coagulation disorders using metal ions

The method according to any one of the preceding claims, characterized in that the blood sample is blood or a blood derived sample such as a blood plasma sample or a blood serum sample.

The method according to any one of the preceding claims, characterized by adding said additional metal ions in the Protein C activation stage.

The method according to any one of the preceding claims, characterized in that said calcium ions are used at a concentration of 0.5 to 20 mmol/L, preferably 1 to 10 mmol/L, of the final assay medium.

The method according to any one of the preceding claims, characterized in that said Mg2+ ions are used in a concentration of 20 μmol/L to 10 mmol/L, preferably 100 μmol/L to 2 mmol/L and, more preferably 200 μmol/L to 1 mmol/L of the final assay medium.

The method according to any one of the preceding claims, characterized in that said Mn2+, Zn2+, Cu2+, Ni2+, Sr2+, and/or Cu+ ions are used in a concentration of 1 μmol/L-2 mmol/L, preferably 5 to 400 μmol/L and, more preferably 10 to 80 μmol/L of the final assay medium.

The method according to any one of the preceding claims, characterized in that the activation of Protein C in said sample precedes or occurs simultaneously with activation of coagulation.

The method according to any one of the preceding claims, characterized in that an activator for Protein C selected from the group comprising Protein C activating snake venom enzymes and thrombin, if desired thrombin in combination with thrombomodulin, is used.

The method according to any one of the preceding claims, characterized in that a recombinant activator for Protein C is used.

The method according to any one of the preceding claims, characterized by using as suitable coagulation activators for the intrinsic pathway compositions comprising

• (a) phospholipid(s) and
• (b) contact activators and/or activated Factors IX, XII or XI or a reagent which generates activated factors IX, XII or XI in vitro.

The method according to any one of the preceding claims, characterized by using native or recombinant human or non-human tissue factor (thromboplastin) from human or non-human species with or without Factor VII/VIIa, or (native or recombinant human or non-human) Factor VIIa and phospholipids as a suitable coagulation activator for the extrinsic pathway.

The method according to any one of claims 1, 3 to 6, 8, 19, 21 and 22, characterized by using in the at least partial activation of coagulation according to the intrinsic, extrinsic or common pathway phospholipid(s) selected from synthetic phospholipids, purified phospholipids or a mixture thereof, or crude extracts of biological sources, specifically extracts from brain, platelets, placenta, egg yolk or from soybeans.

The method according to any one of the preceding claims, characterized by using exogenous activated Factor X, or exogenous Factor X and an exogenous activator for Factor X, or an exogenous activator for endogenous Factor X as a suitable coagulation activator for the common pathway.

The method according to any one of the preceding claims, characterized in that components of the Protein C anticoagulant pathway are added to the reaction medium to compensate for variable functional levels of such components in the sample.

The method according to any one of the preceding claims, characterized in that a fibrin polymerization inhibitor, such as Gly-Pro-Arg-Pro is added to the reaction medium.

The method according to any one of the preceding claims, characterized in that coagulation factors selected from the group comprising Factor VIII/Factor VIIIa, Factor IX, Factor X and prothrombin are added to the reaction medium.

The method according to any one of the preceding claims, characterized in that the coagulation factors used are selected from human or non-human sources or being produced by recombinant technology as wild-type proteins or as modified protein sequences to provide the suitable functional property.

The method according to any one of the preceding claims, characterized by using an exogenous synthetic substrate for either Factor Xa or thrombin in the reaction mixture.