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.

Kit Contains:

  • 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)
Who can benefit from using chromogenic factor VIII assays?
Clinicians or researchers interested in determining FVIII levels in patients with hemophilia A or with elevated factor VIII levels, which may be associated with thrombotic risk. Also, manufacturers interested in determining factor VIII potencies. The chromogenic FVIII activity method has been selected as the reference method by the EP for potency estimation of VIII concentrates, and it is a valuable tool for diagnosis an monitoring of hemophilia and for thrombophilia screening.
What should I do for standards and controls for Factor VIII?
DiaPharma has calibrators, normal and abnormal controls available. Clinicians can also use their own pooled normal plasma; however, it must be calibrated against an international standard to be sure what concentration the plasma has. If the normal plasma does not contain exactly 1 IU/ml ( which corresponds to 100%) factor VIII, the values of the standards must be recalculated in order to obtain a correct factor potency assignment. Reference plasma is available from the National Institute for Biological Standards and Controls (NIBSC). The calibration should be performed by running complete standard curves on two or more independent occasions. Industrial manufacturers, in contrast, use for their standard Mega-2, which is a compilation of factor VIII concentrate.
What are the latest recommendations for measuring potencies of high purity factor VIII concentrates?
  1. A concentrate standard should be used in lieu of plasma standards.
  2. Sample and standard concentrates should be pre-diluted in Factor VIII deficient plasma
  3. All assay buffers should contain 1% (10 mg/ml) of high purity albumin, either bovine or human.
  4. The chromogenic method is the reference method.
What are FVIII inhibitors, and how can I measure them?
FVIII inhibitors appear in about 25% of severe hemophilia A patients. They are most common in patients with inversions, large deletions and nonsense mutations. Inhibitor titer varies 1000-fold; high titer seriously impairs treatment efficacy. Inhibitors can be transient or persistent. and appear irrespective of which FVIII concentrate is selected. High titer patients are treated with high doses of human or porcine concentrate. View the FVIII product pages for kits and assays for FVIII Inhibitor measurements.
What is the function of von Willebrand Factor? Briefly describe von Willebrand disease.
von Willebrand factor (vWf) is a glue-like adhesive protein that is responsible for the adhesion of platelets to damaged vascular endothelium. It also carries and protects factor VIII. von Willebrand disease is a hereditary bleeding disorder caused by moderate-to-severe factor VIII deficiency and low-levels of factor VIII-related antigen (substances necessary for blood clotting). Additionally, there is insufficient von Willebrand factor which also helps blood clot. The von Willebrand factor helps platelets to stick to the blood vessel wall and to each other, which is necessary for normal blood clotting.

Visit the vWF product pages for available kits to measure vWF antigen, activity, and collagen binding.

Protein concentrations in plasma
Component Molecular
Weight kDa
Fibrinogen 330 3000 9
Prothrombin 72 150 2
Factor V 330 20 0.05
Factor VII 50 0.5 0.01
Factor VIII 330 0.1 0.0003
Factor IX 56 5 0.09
Factor X 59 8 0.13
Factor XI 160 5 0.03
Factor XII 80 30 0.4
Factor XIII 320 10 0.03
Protein C 62 4 0.06
Protein S 70 10 (free) 0.14
Protein Z 62 2 0.03
Prekallikrein 86 50 0.6
HMW kininogen 120 70 0.6
Fibronectin 450 300 0.7
Plasminogen 92 200 2
t-PA 60 0.005 0.0001
Urokinase 53 0.004 0.0001
Antithrombin 58 145 2.5
Heparin Cofactor II 66 80 1.2
Plasmin Inhibitor 63 60 1
Protein C Inhibitor 57 4 0.07
α2-Macroglobulin 725 2000 3