DiaPharma Group, Inc.

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For Research use Only

Intended Use

A chromogenic kit for the photometric determination of factor VII activity in human plasma such as when identifying elevated levels of Factor VII or factor VII deficiency and monitoring of patients on replacement therapy. Not affected by preactivation of factor VII.

Principle

The method is based on a two-stage principle. In stage one Factor X is activated to FXa via the extrinsic pathway (FVII-thromboplastin). Factor VII is completely converted to FVIIa during this process and accordingly there is no interference in the assay of preactivated FVII. In stage two the generated Factor Xa hydrolyses the chromogenic substrate S-2765 thus liberating the chromophoric group, pNA. The colour is then read photometrically at 405 nm. The generated Factor Xa and thus the intensity of colour is proportional to the FVII activity in the sample.

Reagents -- Composition, Packaging, Storage and Stability

1. S-2765 1 vial. Chromogenic substrate (N-a-Cbo-D-Arg-Gly-Arg-pNA), (8 mg) with mannitol added (bulking agent). Reconstituted substrate is stable for 6 months at 2-8°C.
2. Bovine Serum Albumin 1 vial. BSA 20%. The opened vial is stable for 1 week at 2-8°C.
3. Buffer stock solution 1 vial. Tris 1.0 mol/L, pH 7.4. The stock solution is stable for 2 months at 2-8°C. Tris-BSA working buffer is stable for 5 days at 2-8°C.
4. Factor X 1vial. Bovine Factor X, 3.1 IU. Reconstituted Factor X is stable for one week at 2-8°C.
5. CaCl2 1 vial. Calcium chloride solution, 40 mmol/L. Stable at 2-8°C until the expiry date printed on the label.
6. Thromboplastin 1 vial. Human placenta thromboplastin. Reconstituted thromboplastin is stable for 1 month at 2-8°C.

When kept at 2-8°C the sealed reagents are stable until the expiry date printed on the label. Contamination by microorganisms should be avoided once the vials are opened.

Reagents not provided

• Normal human plasma for calibration, which should be calibrated against an International Standard. Blood samples are taken from at least 20 healthy donors. 10-30 mL of citrated blood (9 volumes blood and 1 volume 0.1 mol/L sodium citrate) are taken from each donor. The first 3-5 mL of blood is discarded. Plasma is prepared by centrifugation at 2000xg for 20 minutes. Equal amounts of plasma from the donors are mixed and dispensed in small volumes and frozen immediately. The normal plasma is stable for one year at -70°C or for three months at -20°C. Avoid refreezing. Thaw rapidly at 37°C just before use.
• Acetic acid 20% or citric acid 2%.
• Sterile water

Materials required but not provided

• Photometer, 405 nm (and 490 nm for the microplate procedure)
• Heat incubator 37°C ±0.2°C. Verify that the incubator provides an even temperature distribution within these specifications.
• Calibrated pipettes with an accuracy of 1% or better.
• Plastic test tubes
• Semi-micro cuvettes
• Vortex mixer
• Sample diluter/dispenser
• Stopwatch
• Centrifuge, 2000xg

Number of determinations:
Test tube: 30
Microplate: 120
Automated: up to 120