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CHROMOGENIC ASSAYS & RESEARCH METHODS FOR FACTOR VIII MEASUREMENT

Chromogenix Coamatic^® Factor VIII (cat # K822585)

Chromogenix Coatest^® SP Factor VIII (cat# K824086)

Chromogenix Coatest^® SP Factor VIII:C/4 (cat# K824084)

Chromogenix Coamatic^® Factor VIII Elevated Factor VIII Method*

METHOD

Chromogenic Assay

DESCRIPTIONS

Chromogenix Coamatic Factor VIII

A chromogenic kit for the determination of factor VIII activity in human plasma, blood fractions and purified preparations. 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.

Chromogenix Coatest SP Factor VIII

Coatest SP Factor VIII replaces the obsolete Coatest Factor Viii kit (K821033), the classic chromogenic kit for the in vitro diagnostic determination of factor VIII activity in human plasma, blood fractions and purified preparations. The SP Factor VIII kit has two measuring ranges plus validated and documented protocols for a wide range of instruments. The SP kit is the same as the classic FVIII kit with changes to substrate, buffer and phospholipid components.

Chromogenix Coatest SP4 Factor VIII

The Coatest SP4 Factor VIII kit replaces the obsolete Coatest Factor VIII:C/4 (K824094) with same component changes as above and is suitable for low level volume testing.

Chromogenix Coamatic Factor VIII Elevated Factor VIII Method*

A research method for the determination of elevated levels of factor VIII activity in plasma.

REAGENTS AND THEIR STABILITY WHEN OPENED

REAGENT

VIALS

STABILITY

TEMP

Coatest
FVIII**

X

X

6 months

1 month

2-8°C

2-8°C

X

X

X

1 vial

4 vials

2 vials

1 month

12 hours

1 month

12 hours

1 day

1 month

-20 °C

2-8°C

-20 °C

2-8°C

2-8°C

70°C

X

X

X

X

X

 X

 X

X

X

X

X

X

X

X

X

X

X

X

X

MEASUREMENT PRINCIPLE

Factor VIII acts as an enzymatic cofactor of factor IXa during the activation of factor X to factor Xa in the presence of calcium ions and phospholipids. Factor Xa hydrolyses the chromogenic substrate S-2765 thus liberating the chromophore pNA. The color is then read spectrophotometrically at 405 nm. The generated factor Xa and thus the intensity of color is proportional to the factor VIII activity in the sample. Thrombin, that is contained in the factor reagent, brings about a rapid and complete activation of the FVIII present in the sample.

NUMBER OF DETERMINATIONS

PRODUCT

TEST TUBE

MICROPLATE

AUTOMATED

Coamatic Factor VIII

30

120

up to 100

Coatest SP Factor VIII

50

200

up to 200

Coatest SP4 Factor VIII

4 x 15

200

up to 200

ARTICLES & ABSTRACTS

Hemophilia A mutations associated with 1-stage/2-stage activity discrepancy disrupt protein-protein interactions within the triplicated A domains of thrombin-activated factor VIIIa.

Pipe SW, Saenko EL, Eickhorst AN, Kemball-Cook G, Kaufman RJ.

Blood. 2001 Feb 1;97(3):685-91.

PMID: 11157485

Stability and sterility of a recombinant factor VIII concentrate prepared for continuous infusion administration.

Belgaumi AF, Patrick CC, Deitcher SR.

Am J Hematol. 1999 Sep;62(1):13-8.

PMID: 10467271

Mild hemophilia A caused by increased rate of factor VIII A2 subunit dissociation: evidence for nonproteolytic inactivation of factor VIIIa in vivo.

Pipe SW, Eickhorst AN, McKinley SH, Saenko EL, Kaufman RJ.

Blood. 1999 Jan 1;93(1):176-83.

PMID: 9864159

Chandler WL et al. Comparison of Three Methods for Measuring Factor VIII Levels in Plasma.

American Journal of Clinical Pathology

2003;120:34-39.

Sustained expression of human factor VIII in mice using a parvovirus-based vector.

Chao H, Mao L, Bruce AT, Walsh CE.

Blood. 2000 Mar 1;95(5):1594-9.

PMID: 10688813

Human factor VIII can be packaged and functionally expressed in an adeno-associated virus background: applicability to haemophilia A gene therapy.

Gnatenko DV, Saenko EL, Jesty J, Cao LX, Hearing P, Bahou WF.

Br J Haematol. 1999 Jan;104(1):27-36.

PMID: 10027708

Hemophilia A mutations associated with 1-stage/2-stage activity discrepancy disrupt protein-protein interactions within the triplicated A domains of thrombin-activated factor VIIIa.

Pipe SW, Saenko EL, Eickhorst AN, Kemball-Cook G, Kaufman RJ.

Blood. 2001 Feb 1;97(3):685-91.

PMID: 11157485

Stability and sterility of a recombinant factor VIII concentrate prepared for continuous infusion administration.

Belgaumi AF, Patrick CC, Deitcher SR.

Am J Hematol. 1999 Sep;62(1):13-8.

PMID: 10467271

Mild hemophilia A caused by increased rate of factor VIII A2 subunit dissociation: evidence for nonproteolytic inactivation of factor VIIIa in vivo.

Pipe SW, Eickhorst AN, McKinley SH, Saenko EL, Kaufman RJ.

Blood. 1999 Jan 1;93(1):176-83.

PMID: 9864159

Pharmacokinetic in vivo comparison using 1-stage and chromogenic substrate assays with two formulations of Hemofil-M.

Lee C, Barrowcliffe T, Bray G, Gomperts E, Hubbard A, Kemball-Cook G, Lilley P, Owens D, Von Tilberg L, Pasi J.

Thromb Haemost. 1996 Dec;76(6):950-6.

PMID: 8972016

PMID (PubMed - indexed for MEDLINE)

DETERMINATION OF ELEVATED LEVELS OF FACTOR VIII ACTIVITY

Background

Historically, factor VIII (FVIII) has always been associated with bleeding, since the well known pathological condition Haemophilia A is a consequence of very low plasma levels of FVIII. On the other hand, FVIII is also an acute phase reactant, and hence elevated levels are associated with conditions such as trauma, infection and exercise. Common to many other coagulation factors, FVIII also rises during pregnancy. FVIII is a key procoagulant factor and recent studies have shown the association between elevated levels of FVIII activity and an increased risk of venous and seemingly also arterial thrombosis (1-4).

A concomitant increase was also noticed for von Willebrand factor (1-3). Importantly, FVIII activity shows a high correlation to FVIII antigen (FVIII:Ag), thereby attributing the increased activity to an increased FVIII synthesis (2, 5). So far the increased plasma FVIII:Ag has not been linked to any polymorphism of the FVIII gene promoter 5 , but the search for a genetic contribution is still under investigation. FVIII activity in thrombotic patients is often above 1.5 IU/ml and might reach levels of 4-5 IU/ml (2), sometimes in connection with highly inflammatory conditions.

Therefore, a specific adaptation of Coamatic Factor VIII has been developed to allow accurate determination of elevated FVIII activity. The advantages in using a chromogenic method as compared to one-stage clotting methods are numerous. In particular the chromogenic method is not sensitive to preactivation of FVIII (6), thereby avoiding overestimation of FVIII activity.

Furthermore, due to its linear dose-response, it has a higher resolution at elevated levels and also a high precision. These features make Coamatic Factor VIII ideal as a tool for thrombophilia screening in addition to its established use for diagnosis of hemophilia and FVIII potency estimation of concentrates.

The applications of the Coamatic Factor VIII kit are currently referred to a low assay range and to a normal assay range. The upper measuring limit using the procedure for the normal assay range is 1.42 IU/ml for the microplate method and 1 IU/ml for the ACL method. The determination of FVIII activities higher than these limits, can be performed by pre-diluting the plasma samples 1:4 and assaying the diluted samples following the protocol described for the normal assay range but restricting this range to 0-1 IU/ml.
The results should be multiplied by 4 to obtain the final value of FVIII activity.

• Pre-dilute the samples using the buffer contained in the Coamatic Factor VIII kit as follows:
  1 vol plasma sample + 3 vol diluted buffer
• Dilute further as detailed in the package insert
• Follow the instructions contained in the Coamatic Factor VIII package insert
  (microplate procedure) or in the instrument application sheet (automated instruments)

eFVIII - MICROPLATE METHOD

Reagent preparation
Factor reagent: 3.0 ml of sterile water
Substrate: 6.0 ml of sterile water
Buffer: dilute 1:10 with sterile water

Standard curve
The standard curve 0-1 IU/ml is prepared by using a human normal plasma calibrated against an International Standard for plasma FVIII. In case the normal plasma does not contain exactly 1 IU/ml FVIII, the values of the standard must be recalculated accordingly.

Sample Dilution

1. Pre-dilute the sample by mixing 1 vol plasma with 3 vol of Coamatic Factor VIII Buffer
2. Dilute further as follows: 25 µl Sample + 2000 µl Buffer

Assay procedure

Read the absorbance at 405 nm, using a reference wavelength of 490 nm.

Fig. 1. Standard curve with the microplate method.

eFVIII - ACL METHOD

This method is applicable to the ACL™200/300/3000/6000/7000.

Reagent preparation
Factor reagent: 3.0 ml of sterile water
Substrate: 5.25 ml of sterile water
Buffer: dilute 1:10 with sterile water

Standard curve
The standard curve is prepared by using a human normal plasma calibrated against an International Standard for plasma FVIII.
Dilute the standard as follows: 25 µl plasma + 2000 µl buffer

Sample Dilution

1. Pre-dilute the sample by mixing 1 vol plasma with 3 vol of Coamatic Factor VIII Buffer
2. Dilute further as follows: Sample 25 µl Buffer 2000 µl

Assay procedure
Select the test Plasminogen (channel).
Place diluted normal plasma in POOL position.
Place buffer working solution in DIL position.
Place factor reagent in position 2.
Place substrate in position 3.
Place sample cups with diluted plasmas.

Fig. 2. Standard curve with the ACL method.

eFVIII - MEASURING RANGE

With pre-dilution of the sample the measuring range is 1 – 4 IU/ml with both the microplate and the ACL method.

eFVIII - RESULTS

The evaluation of Coamatic Factor VIII with samples from thrombotic patients has been performed both with the microplate and the ACL applications. The standard curves are shown in figures 1 and 2 respectively. The upper limit of the standard curve is 1 IU/ml in both methods resulting in an upper measurement limit of 4 IU/ml, with plasma samples diluted 1:4. The precision of the method has been evaluated by using plasma samples diluted according to the protocol described above.

The FVIII activity of 130 patient samples has been determined with Coamatic Factor VIII on ACL, by pre-diluting or not the plasma samples. The samples have been obtained from patients about three months after the thrombotic episode. The following results were obtained from linear regression analysis (figure 3):
Slope = 1.52
Intercept = -0.57
R = 0.96
Range (x) = 0.45 – 3.28 IU/ml FVIII
Range (y) = 0.33 – 4.50 IU/ml FVIII

Fig. 3. Comparison of FVIII activities obtained with and without sample pre-dilution using Coamatic Factor VIII.

For FVIII activities higher than 1 IU/ml, the samples can be under-estimated if the pre-dilution is not performed.

Coamatic Factor VIII has been compared with a one-stage clotting method on the ACL analyser. For the Coamatic Factor VIII assay, the samples were pre-diluted 1:4 as recommended in the protocol described above. For the clotting method the plasma samples were pre-diluted 1:4 (with 0.05 mol/l imidazol, 0.1 mol/l NaCl, pH 7.3; buffer recommended by the clotting reagent manufacturer) followed by the prescribed sample dilution 1:5. 71 plasma samples from thrombotic patients were analysed. The results are shown in figure 4.

Fig. 4. Comparison of FVIII activities obtained by a clotting assay and Coamatic Factor VIII.

The following results were obtained from linear regression analysis:
Slope = 1.28
Intercept = -0.43
R = 0.92
Range (x) = 0.50 – 2.32 IU/ml FVIII
Range (y) = 0.18 – 2.51 IU/ml FVIII

eFVIII - CONCLUSIONS

The results described here represent a preliminary evaluation of Coamatic Factor VIII applied for the screening of samples from thrombotic patients. From the population of samples tested, about 25% had a FVIII activity higher than 1.4 IU/ml, thus confirming earlier published data 1,2 . These results have been obtained by a simple modification of the existing applications and protocols, consisting in the pre-dilution 1:4 of the plasma samples.

Coamatic Factor VIII is a kit suitable for use on a number of automated instruments as well as on microplates. The data presented here show its applicability on the ACL instrument for determination of elevated FVIII activity. In case the pre-dilution is done manually, the current application notes for automated instruments can then be adhered to, with the only exception of restricting the assay range to 0-1 IU/ml. Indeed, some instruments offer the possibility of also performing the pre-dilution step.

eFVIII - BIBLOGRAPHY

1. Koster T, Blann AD, Briët E, Vanderbroucke JP, Rosendaal FR. Role of clotting factor VIII in effect of von Willebrand factor on occurrence of deep-vein thrombosis. Lancet 345; 152-155 (1995).

2. O’Donnell J, Tuddenham EGD, Manning R, Kemball-Cook G, Johnson D, Laffan D. High prevalence of elevated factor VIII levels in patients referred for thrombophilia screening: role of increased synthesis and relationship to the acute phase reaction. Thromb Haemost 77; 825-828 (1997).
3. Gorog DA, Rkhit R, Parums D, Laffan M, Davies GJ. Raised factor VIII is associated with coronary thrombotic events. Heart 80; 415-417 (1998).
4. Meade TW, Mellows S, Brozovic M, Miller GJ, Chakrabarti RR, North WRS, Haines AP, Stirling Y, Imeson JD, Thomsom SG. Haemostatic function and ischaemic heart disease: principal results of the Northwick Park Heart study. Lancet ii: 533-537 (1986).
5. Mansvelt EPG, Laffan M, McVey JH, Tuddenham EGD. Analysis of the F8 gene in individuals with high plasma factor VIII:C levels and associated venous thrombosis. Thromb Haemost 80; 561-565 (1998).

6. Rosén S, Andersson M, Blombäck U, Hägglund U, Larrieu MJ, Wolf M, Boyer C, Rothschild C, Nilsson IM, Sjörin E, Vinazzer H. Clinical application of a chromogenic substrate method for determination of factor VIII activity.

eFVIII - ADDITIONAL PUBLICATIONS

• Chandler WL et al. Comparison of Three Methods for Measuring Factor VIII Levels in Plasma. American Journal of Clinical Pathology 120, 34-39 (2003).

• Kraaijenhaagen RA et al. High plasma concentration of factor VIII:C is a mjor risk factor for venous thromboembolism. Thromb Haemost 83, 5-9 (2000).
• O'Donnell J et al. Elevation of FVIII:C in venous thromboembolism is persistent and independent of the acute phase response. Thromb Haemost 83, 10-13 (2000).
• Schambeck CM et al. Venous thromboembolism and associated high plasma factor VIII levels : Linked to Cytomegalovirus infection? Thromb Haemost 83, 510-511 (2000).

• Rosendaal FR. High levels of factor VIII and venous thrombosis. Thromb Haemost 83, 1-2 (2000).

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