Chromogenix Coatest® SP4 Factor VIII is a chromogenic assay kit for the in vitro diagnostic photometric determination of factor VIII activity in citrated plasma.

Reagents, 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.
FIXa, Ca2+, phospholipid
Factor XFXa
FVIII
FXa
S-2765™Peptide + pNA

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.

Factor VIII is a non-enzymatic plasma protein that is essential for normal blood coagulation. The deficiency of factor VIII activity in humans is associated with a congenital bleeding disorder, called hemophilia A, which affects about 1 in 5000 males. Hemophilia A patients are treated with factor VIII concentrate for maintenance of normal hemostasis but regrettably prophylactic treatment is not in general use worldwide. During later years recombinant factor VIII has been approved for therapeutic use, which minimizes the risk of viral transmission. There is now also growing evidence that elevated factor VIII activity is a risk factor for thrombosis. Hence, factor VIII levels are of importance to measure not only for diagnosing and monitoring hemophilia A but also for thrombophilia investigations. With the advent of chromogenic substrate technology accurate and sensitive methods are available for quality control and for the clinical coagulation laboratory. It is the purpose of this monograph to present an overview of biochemical and clinical data on factor VIII and to provide comprehensive information on methodological aspects and on the use of the Coamatic® and Coatest® Factor VIII kits.


Read more about chromogenic determination of Factor VIII…

How do factor V and VIII levels affect the Chromogenix Coatest® APC Resistance tests?
A study by Colucci et al. (Thromb. Haemost. 1994, 72:987 – 988) using the classical APTT method (no FV-deficient plasma pre-dilution) showed that changes in FV levels between 12.5-100% did not modify the response to APC. Other experience has shown that the Coatest® APC Resistance V test may provide ratio values approximately 0.3 units below the median level, which can be fairly close to the cut-off value, when FV levels are 0-40%. This may be explained by the fact that although there is often no abnormal bleeding tendency in heterzygotes with factor V deficiency, prolonged PT and APTT times are observed (ref: Sartori et al. Familial association of hypoplasminogenemia and heterozygous factor V deficiency. Clin Appl Thromb Hemost. 1999; 5(4): 277-281; Salooja N et al. Severe factor V deficiency and neonatal intracranial haemorrhage: a case report. Haemophilia. 2000; 6(1): 44-46. One might also see a slightly decreased ratio in patients with severe liver disease.

According to Chromogenix, FVIII samples above 1.8 IU/ml may result in a reduction of the APC ratio of approximately 0.2 units, although the actual correlation between FVIII activity and APC ratio appears to be weak. When sampling, therefore, the patient should be at rest in order to decrease the FVIII level due to stress.

 
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.

Does the Chromogenix Coatest® SP FVIII detect animal plasma?
Yes, it will detect FVIII activity in dog, cat, horse, rabbit, and mouse plasma.
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.
Sometimes when I run the Chromogenix Coamatic® FVIII assay, I see an upward drift in my activity from the first to the last samples. Why is this?
Thrombin, 1 NIH unit, is included in the factor reagent. It has been shown that thrombin can activate FX, and over time you will see an increase of up to 5% activity. If you are doing numerous samples at once on an automated instrument, you might want to consider Coatest® FVIII.
Is it OK to use glass tubes for the Chromogenix Factor VIII assay?
No, glass surfaces may interfere with the generation of FXa. Use plastic tubes for the manual method.
If thrombin is formed, will it hydrolyze the substrate S-2765™ when using Chromogenix Coamatic®, Coatest® SP, or Coatest® SP4 FVIII kits?
No, hydrolysis of S-2765™ by thrombin is prevented by the addition of the synthetic thrombin inhibitor I-2581.
The standard pre-dilutions for the Coatest® SP FVIII assay dont make sense to me. For example, to get 100%, I dilute 100 ml plasma with 50 ml buffer, but to get my 50% standard, I dilute 100 ml plasma with 200 ml buffer. Why?
The key is that the plasma that does not have a pre-dilution corresponds to 150%, not 100%. One way to look at it is that since 100 is 67% of 150, then the plasma must be diluted 67%, which corresponds to 100 ml plasma with 50 ml buffer (100 plasma / 150 total volume = 0.67). This process can be followed for all of the standard pre-dilutions.
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 is the difference between Chromogenix Coamatic® FVIII, Coatest® SP FVIII, and Coatest® SP FVIII?
All of these kits measure the same thing — factor VIII activity in plasma and potency estimation of FVIII concentrates. Coamatic® FVIII includes the FXa substrate S-2765™, and the factor reagent FIXa + FX and thrombin colyophilized with CaCl2 and phospholipid. The Coatest® SP FVIII includes the FXa substrate S-2765™, and the FIXa + FX reagent, CaCl2, and phospholipid are all separate components in the kit. Incubation times are a little longer, and the reagent prep is less simple. Coatest® SP4 FVIII is exactly the same as Coatest® SP FVIII, but the FIXa + FX reagent is divided into 4 vials of 1.8 U (reconstituted with 3 ml) instead of 1 vial of 6 U (reconstituted with 10 ml).
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 (NBSB). 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 is the measurement principle behind the FVIII chromogenic kits?
First, factor VIII circulates in the plasma bound to von Willebrand factor (vWf). Thrombin cleaves and activates factor VIII and releases vWf. The vWf is then free to bind to ruptured endothelial cell surfaces where it activates platelet aggregation. The released FVIIIa acts as a cofactor of factor IXa to generate factor Xa. In the presence of Ca2+ and phospholipids, FX is activated to FXa by FIXa (look at the coagulation cascade for a better understanding). Since FVIIIa is a cofactor to FIXa, it greatly stimulates the reaction. By using optimal amounts of Ca2+, phospholipid, and FIXa, and an excess of FX, the rate of activation of FX is linearly related to the amount of FVIII. FXa hydrolyses the chromogenic substrate S-2765™ which releases the chromophoric group pNA. The color is read at 405 nm, and generated FXa and thus the intensity of color, is proportional to the FVIII activity in the sample.
What are the latest recommendations for measuring potentcies 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.
Why is thrombin added in the Chromogenix Coamatic® FVIII factor reagent?
Thrombin activates FX, and this happens when the reagent is reconstituted. An incubation step is required in the Chromogenix Coatest® FVIII kit.
Can I use cryoprecipitate samples with the Chromogenix Coatest® FVIII kits?
Yes. Predilute cryoprecipitate in the Coatest® FVIII kit buffer to 1 IU/ml, then follow the kit insert. Somewhat larger dilutions than described in the kit may be necessary with some concentrations.
Protein concentrations in plasma
ComponentMolecular
Weight kDa
Plasma
Concentration
mg/l
Plasma
Concentration
μmol/l
Fibrinogen33030009
Prothrombin721502
Factor V330200.05
Factor VII500.50.01
Factor VIII3300.10.0003
Factor IX5650.09
Factor X5980.13
Factor XI16050.03
Factor XII80300.4
Factor XIII320100.03
Protein C6240.06
Protein S7010 (free)0.14
Protein Z6220.03
Prekallikrein86500.6
HMW kininogen120700.6
Fibronectin4503000.7
Plasminogen922002
t-PA600.0050.0001
Urokinase530.0040.0001
Antithrombin581452.5
Heparin Cofactor II66801.2
Plasmin Inhibitor63601
Protein C Inhibitor5740.07
α2-Macroglobulin72520003