For in vitro research use only

Functional assay proposed for the measurement of microparticles' procoagulant activity in human plasma, in vitro, using automated or manual method.

APPLICATIONS

Assay of microparticles in contexts such as cardiovascular diseases, malignancy, infectious or inflammatory pathology.

ASSAY PRINCIPLE

The diluted assayed plasma sample, supplemented with calcium, Factor Xa and thrombin inhibitors, is introduced into one of the microplate wells coated with Streptavidine and biotinylated Annexin V, then incubated. Following a washing step, the Factor Xa-Va mixture containing calcium, then the purified prothrombin, are introduced. When present in the tested sample, microparticles bind to Annexin V and expose their phospholipids surface, thus allowing to FXa-FVa, in presence of calcium, to activate prothrombin into thrombin. The phospholipids concentration is then the limiting factor. There is a direct relationship between the phospholipids concentration and the amount of thrombin generation, which is measured via its specific activity on the thrombin substrate. The reaction is stopped with 2% Citric Acid and Absorbance is measured at 405 nm (A405).

Functional Method

Functional Method

Immunological Method

Calibration Curve

REAGENTS -- PREPARATION, STORAGE AND STABILITY

In their original packaging box, before use, when stored at 2-8°C, the unopened reagents are stable until the expiration date printed on the box.

1. Micro ELISA plate (COAT) containing 12 strips of 8 wells, coated with Streptavidine and biotinylated Annexin V, then stabilized; the plate is packed in an aluminium pouch hermetically sealed in presence of a desiccant. Open the plastic pouch and take off the required amount of 8 well strips for the test series. When out of the pouch, the strips must be used within 30 minutes. Unused strips can be stored at 2-8°C for 4 weeks in their original aluminium pouch, in presence of the desiccant, hermetically closed and protected from any moisture, and stored in the provided microplate storage bag (minigrip).
2. SD-MP (Sample Diluent): 2 vials of 50 ml containing FIIa and FXa inhibitors, ready to use. When open, it can be used for 4 weeks, stored at 2-8°C, and provided that any bacterial contamination is avoided during use. This reagent contains 0.05% Kathon CG.
3. R1: 2 vials of Bovine FXa-FVa mixture, containing calcium, lyophilized (to be reconstituted with 6 ml distilled water). Reconstitute each vial with exactly 6 ml of distilled water; Shake thoroughly until complete dissolution of the content (vortex). Incubate at room temperature (18-25°C) for 30 min, and at least for 15 min at 37°C before use, while shaking the vial from time to time. Homogenise the content before each use. Following reconstitution, the reagent is stable for 24 hours at 2-8°C, 8 hours at room temperature (18-25°C), or 2 months at -20°C or below.
4. R2: 2 vials of purified human Prothrombin, lyophilized (to be reconstituted with 3 ml distilled water). Reconstitute each vial with exactly 3 ml of distilled water; Shake thoroughly until complete dissolution of the content (vortex). Incubate at room temperature (18-25°C) for 30 min, and at least for 15 min at 37°C before use, while shaking the vial from time to time. Homogenise the content before each use. Following reconstitution, the reagent is stable for 24 hours at 2-8°C, 8 hours at room temperature (18-25°C), or 2 months at -20°C or below.
5. R3: 2 vials of Thrombin specific chromogenic substrate (IIa), lyophilized (to be reconstituted with 3 ml distilled water). Reconstitute each vial with exactly 3 ml of distilled water; Shake thoroughly until complete dissolution of the content (vortex). Incubate at room temperature (18-25°C) for 30 min, and at least for 15 min at 37°C before use, while shaking the vial from time to time. Homogenise the content before each use. Following reconstitution, the reagent is stable for 1 month at 2-8°C, 72 hours at room temperature (18-25°C), or 2 months at -20°C or below.
6. Cal: 2 vials of Calibrator, lyophilized, prepared from a washed and lysed platelet concentrate. When restored with 2 ml of Sample Diluent (SD-MP), the ready to use calibrator, with a concentration "C" (in nM) of microparticles, established respectively to an internal standard, is obtained. This concentration is expressed in nM Phosphatidyl Serine (PS) equivalent. Restore each vial with 2 ml Sample Diluent (SD-MP) in order to obtain the ready to use calibrator containing "C" (nM) of microparticles. This concentration is expressed in nM Phosphatidyl Serine (PS) equivalent. Following reconstitution, the calibrator is stable for 24 hours at 2-8°C, 8 hours at room temperature (18-25°C), or 2 months at -20°C or below.
7. CI: 1 vial of 0.5 ml of plasma Control I, lyophilized. Restore each vial with 0.5 ml distilled water. Following reconstitution, the control is stable for 24 hours at 2-8°C, 8 hours at room temperature (18-25°C), or 2 months at -20°C or below.
8. CII: 1 vial of 0.5 ml of plasma Control II, lyophilized. Restore each vial with 0.5 ml distilled water. Following reconstitution, the control is stable for 24 hours at 2-8°C, 8 hours at room temperature (18-25°C), or 2 months at -20°C or below.
9. WS-MP: 1 vial of 50 ml of 10 fold concentrated Wash Solution. If necessary, incubate the vial for 15-30 minutes in a water bath at 37°C until complete dissolution of solids when present. Shake the vial and dilute the amount required 1:10 in distilled water (the 50 ml contained in the vial allow to prepare 500 ml of Wash Solution). The Wash Solution must be stored at 2-8°C in its original vial and used within 8 weeks following opening. The diluted Wash Solution must be used within 7 days, when protected from any contamination and stored at 2-8°C. This reagent contains 0.05% Kathon CG.
10. CA: 1 vial of 6 ml of 2% Citric Acid, stop solution, ready to use.