Intended Use

For the in vitro diagnostic photometric determination of factor VIII activity in citrated plasma.

Measurement principle

In the presence of calcium and phospholipids, factor X is activated to factor Xa by factor IXa. This generation is greatly stimulated by factor VIII, which may be considered as a cofactor in this reaction. By using optimal amounts of Ca2+ and phospholipids and an excess of factors IXa and X, the rate of activation of factor X is solely dependent on the amount of factor VIII. Factor Xa hydrolyses the chromogenic substrate S-2765 thus liberating the chromophoric group, pNA. The color is then read photometrically at 405 nm. The generated factor Xa and thus the intensity of color is proportional to the factor VIII activity in the sample. Hydrolysis of S-2765 by thrombin formed is prevented by the addition of the synthetic thrombin inhibitor, I-2581, together with the substrate.

Reagents -- Composition, Packaging, Storage, Stability, and Microplate/Test Tube Preparation

1. S-2765 15.4 mg + I-2581 1 vial. Chromogenic substrate (N-a-Z-D-Arg-Gly-Arg-pNA), 15.4 mg, synthetic thrombin inhibitor, 0.4 mg, and mannitol added as a bulking agent. Reconstitute with 12.0 mL of sterile water or NCCLS type II water, to obtain a concentration of 2.7 mmol/L. Stability after reconstitution: 3 months at 2-8°C.
2. Factor IXa + factor X 2.7 IU 4 vials. Lyophilized bovine factors IXa and X with bovine albumin added as a stabilizing agent. Reconstitute with 3.0 mL of sterile water or NCCLS type II water. Stability after reconstitution: 12 hours at 2-8°C. The solution can be stored frozen in aliquots at -20°C (or at lower temperature) for 3 months. Do not refreeze.
3. CaCl2 6 ml 1 vial. Calcium chloride solution, 0.025 mol/L Ready to use. Opened vial is stable 3 months at 2-8°C.
4. Buffer, stock solution 20 mL 1 vial. 20 mL concentrated Tris buffer containing NaCl and BSA. Characteristics of tenfold diluted buffer: Tris 0.05 mol/L, pH 7.3, 10 mg/L Ciprofloxacin and 1.0% BSA. Dilute 1:10 (1+9) with sterile water or NCCLS type II water. Prepare a new buffer working solution each day. Once opened the buffer.
5. Phospholipid 2 mL 1 vial. Mixture of highly purified phospholipids and 10 mg/L Ciprofloxacin. Ready to use. Opened vial is stable for 3 months at 2-8°C. Shake gently before use.

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.