Rox Factor VIII is a chromogenic kit for the determination of Factor VIII (FVIII) activity in human plasma and FVIII containing concentrates. This kit is for in vitro research use only and should not be used for patient diagnosis or treatment.
FVIII is a plasma protein of about 230 000 Daltons (230kD), which serves as a cofactor to Factor IXa in its activation of Factor X to Factor Xa. Deficiency of FVIII causes the severe bleeding disorder Hemophilia A. FVIII is stabilized by its binding to von Willebrand Factor (vWF), a multimeric glycoprotein which prolongs the half-life of FVIII in blood circulation.
In the presence of Ca2+ and phospholipids, Factor X is activated to Factor Xa by Factor IXa. This reaction is greatly stimulated by FVIII after activation to FVIIIa by thrombin. By using optimal concentrations of Ca2+, phospholipids and an excess of Factor IXa, Factor X and thrombin, the rate of activation of Factor X is directly related to the amount of FVIII in the sample. Factor Xa hydrolyses the chromogenic Factor Xa substrate, Z-DArg-Gly-Arg-pNA, thus liberating the chromophoric group pNA. The color is read photometrically at 405nm and the generated FXa and thus the intensity of color is proportional to the FVIII activity in the sample.
200 tests per kit in the microplate mode, using 50mL volumes throughout of:
sample/std dilution, Reagent 1, Reagent 2 and FXa Substrate.
- Reagent 1 (2 vials): lyophilized bovine FX and a fibrin polymerization inhibitor.
- Reagent 2 (2 vials): lyophilized human FIIa, human FIXa, calcium chloride and phospholipids.
- FXa Substrate, 6mL (2 vial): Liquid solution of chromogenic FXa substrate (ZD-Arg-Gly-Arg-pNA), 2.5mmol/L, containing a thrombin inhibitor. Contains sodium azide ≤ 0.01% (≤0.1g/L)
- Tris BSA Buffer, Stock Solution, 20mL (1 vial): Liquid stock solution of diluent buffer, containing 10% Bovine Serum Albumin (BSA) and a heparin antagonist. Stock solution contains sodium azide ≤ 0.01% (≤0.1g/L)
- Unsurpassed accuracy and reagent stability
- One range, only one calibration curve is used for the whole measuring range (0-200%).
- Normal sample dilution = 1:60, Samples with activity <5% are diluted 1:15.
- Excellent linearity over entire measuring range, including very low Factor VIII levels.
- Excellent discrimination at low Factor VIII activities.
- Conditions during activation are in agreement with the European Pharmacopoeia chapter 2.7.4 Assay of human coagulation factor VIII
- Stability of Reagents after reconstitution = 72 hours at 2-8°.
- Reagents can be aliquoted and frozen at ≤ -70° in order to limit reagent waste.
- Not sensitive to Emicizumab (due to use of bovine Factor X in Reagent 1)