DiaPharma Group, Inc.

For in vitro diagnostic use

Intended use

A chromogenic kit for the photometric determination of factor VIII activity in plasma, blood fractions and purified preparations, such as when identifying factor VIII deficiency or monitoring patients on replacement therapy as well as for potency estimation of FVIII concentrates. Fulfills the requirements of the European Pharmacopoeia for factor VIII concentrate testing. Co-lyophilization of reagents for convenient handling. Reliable tool for hemophilia classification. No interference from heparin levels up to 1.5 IU/mL. Suitable for determination of elevated factor VIII in thrombophilia screening. The Factor VIII kit has two measuring ranges plus validated and documented protocols for a wide range of instruments.

Background and summary

The Coamatic Factor VIII kit provides reagents and chromogenic methods for determining factor VIII activity in human plasma and in factor VIII concentrates. Factor VIII is a high molecular weight plasma protein which serves as a cofactor to factor IXa in its activation of factor X to Xa.

Deficiency of factor VIII causes a severe bleeding disorder, hemophilia A. The severity of this bleeding disorder is inversely related to the factor VIII concentration. Hemophilia A patients are generally classified according to their factor VIII activity into three categories: <0.01 IU/mL = severe, 0.01-0.04 IU/mL =moderate and 0.05-0.25 IU/mL = mild hemophilia.

Measurement principle

In the presence of calcium ions and phospholipids, factor X is activated to factor Xa by factor IXa. This activation is greatly stimulated by factor VIII which acts as a cofactor in this reaction. By using optimal amounts of Ca2+, phospholipid and factor IXa, and an excess of factor X, the rate of activation of factor X is linearly related to the amount of factor VIII. 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 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 and Stability

1. S-2765 + I-2581 1 vial. Chromogenic substrate (N-a-Z-D-Arg-Gly-Arg-pNA) (7.7 mg) and synthetic thrombin inhibitor (0.2 mg) with mannitol (bulking agent). Stability after reconstitution: 1 month at 2-8°C.
2. Factor reagent 2 vials. Bovine factor IXa (0.3 U) , factor X (2.7 IU) and thrombin (1 NIH-U) colyophilized with CaCl2 (40 mmol) and phospholipid (0.2 mmol). Stability after reconstitution: 12 hours at 2-8°C, 2 weeks at -30°C or 1 month at -70°C. Avoid storage at -20°C.
3. Buffer, stock solution 1 vial. 24 mL concentrated Tris buffer, containing NaCl and BSA. Characteristics of diluted buffer (1:10): Tris 0.025 mol/L, pH 7.9, I = 0.08, 1% BSA. Stability after dilution: 1 month at 2-8°C.

The reagents are not interchangeable between lots.

The sealed reagents are stable at 2-8°C until the expiry date printed on the label. Avoid contamination by microorganisms in the reagents. Warning: Do not use reagents beyond the expiry date printed on the package label. If the substrate solution appears yellow, discard.

Additional reagents

1. Deionized water, filtered through 0.22 mm or NCCLS type II water.
2. Calibration plasma, calibrated against an International Standard
3. Control Plasma Abnormal and Normal, calibrated against an International Standard for Factor VIII
4. Saline (0.9% NaCl).
5. Acetic acid 20% or citric acid 2% (end-point methods).

Material required but not provided

• Spectrophotometer, 405 nm (and 490 nm for microplate procedure)
• Incubator 37°C ±+0.2
• Microplate or semi-micro cuvettes
• Centrifuge, 2000 x g
• Plastic test tubes
• Stopwatch
• Vortex mixer
• Calibrated pipettes
• Linear graph paper

Number of determinations:

• 30 (test tube)
• 120 (microplate)
• up to 100 (automated)