DiaPharma Group, Inc.

THIS PRODUCT PAGE REMAINS ONLINE FOR HISTORICAL PURPOSES - REPLACEMENT: K824086 (824086) CHROMOGENIX COATEST SP FACTOR VIII

Intended Use

For the 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-2222 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-2222 by thrombin formed is prevented by the addition of the synthetic thrombin inhibitor, I-2581, together with the substrate.

Reagents -- Composition, Packaging, Storage and Stability

1. S-2222 20 mg + I-2581 1 vial. Chromogenic substrate (Bz-Ile-Glu(g-OR)-Gly-Arg-pNA), 20 mg and synthetic thrombin inhibitor, 335 µg with mannitol added as a bulking agent. Stability after reconstitution: 6 months at 2-8°C.
2. Factor IXa + factor X 9.2 IU 1 vial. Lyophilized bovine factors IXa and X with bovine albumin added as a stabilizing agent. 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 1 month. Some activity (<10%) may be lost during freezing and thawing. The time needed to freeze the reagent should be as short as possible.
3. CaCl2 6 mL 1 vial. Calcium chloride solution, 0.025 mol/L. Stable at 2-8°C until the expiry date printed on the label.
4. Buffer, stock solution 20 mL 1 vial. Tris 0.05 mol/L, pH 7.3 and 0.2% Bovine albumin. 20 mL concentrated Tris Buffer containing NaCl and BSA. Characteristics of tenfold diluted buffer: Tris 0.05 mol/L, pH 7.3 and 0.2% BSA. Once opened the buffer is stable 1 month at 2-8°C. Prepare a new buffer working solution each day.
5. Phospholipid 2 mL 1 vial. Mixture of highly purified phospholipids. Opened vial is stable for 1 month 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.

Reagents required but not provided

Deionized water, filtered through 0.22 mm or NCCLS type II water.

Acetic acid 20% or citric acid 2%.

Normal human plasma. Prepare a normal plasma pool using the same procedure as for the plasma samples (see SPECIMEN COLLECTION). Blood samples are taken from at least 20 healthy donors. The plasma pool should be calibrated against the International Reference Plasma for FVIII Related Activities (available from National Institute for Biological Standards and Controls (NIBSC), London, UK).

Material required but not provided

• Photometer, 405 nm (and 490 nm for microplate procedure)
• Heat incubator 37°C ±0.2°C
• Semi-micro cuvettes
• Centrifuge, 2000xg
• Plastic test tubes
• Stopwatch
• Vortex mixer
• Calibrated pipettes