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A Practical Guide to Laboratory Haemostasis

 

2-Stage & Chromogenic Factor Assays



Introduction

The 2-stage assay is rarely performed today but remains an important test. The 2-stage assay has been replaced in many cases by a chromogenic assay. If we consider FVIII assays, the 1-stage factor FVIII assay is relatively simple to perform and accurate. However, it does have limitations including susceptibility to interference from preactivation of factor VIII or anti-phospholipid antibodies as well as misleading results when assaying recombinant factor VIII. In addition some F8 mutations can lead to discrepant 1-stage/2-stage [chromogenic] FVIII assay results.
Two main alternatives to the one stage assay exist, both based around an initial step to produce factor Xa in a quantity proportional to the amount of factor VIII present and a second step to assay the amount of factor Xa and so deduce the amount of factor VIII present. These two alternatives, the 2-stage APTT based assay and the chromogenic assay are discussed below. Their methods are discussed separately followed by comments relevant to both.

Principles & Methodology
2-Stage FVIII Assay

The assay is similar to the chromogenic  assay in that it involves an incubation step for production of FXa and a second stage to determine the amount of FXa produced.
The 2-stage FVIII assay requires:

Component Explanation
Activated serum This provides factors IX, X, XIa to initiate coagulation in the patient sample. Commercially available or prepared by incubating whole blood in glass, centrifuging and then removing the serum.
Factor V Commercially available, usually bovine. Factor V is required as a cofactor for the initial coagulation reaction
Adsorbed patient plasma Adsorption with Al(OH)3 removes factors II, VII, IX and X. This prevents progression of the first stage beyond assembly of the prothrombinase complex
Normal plasma To supply prothrombin and fibrinogen so that coagulation can proceed to clot formation
CaCl2 and phospholipid The reaction is dependent on calcium ions for factor activity and phospholipid surfaces to facilitate factor interaction

Adsorbed patient's plasma is mixed with activated serum, factor V, calcium and phospholipid to initiate coagulation and generate factor Xa. After a fixed period of incubation an aliquot of the mixture is added to an aliquot of normal plasma and the time to clot formation measured. Clotting times are plotted on double log paper and from which the factor VIII level in the patient sample can be derived.
The patient’s plasma is adsorbed to remove prothrombin and so prevent clot formation when the coagulation cascade is initiated. Coagulation is initiated by addition of factor XIa and factor X and V are provided in excess. The factor VIII level is the rate limiting step in the formation of factor Xa. This is the first stage of the assay. In the second stage, a sample of the 1st stage mixture is added to normal plasma and the time to clot formation recorded. Since the clotting time will be dependent on the factor Xa level in the sample from the first stage and the factor Xa level  is, in turn, proportional to the concentration of factor VIII in the patient’s plasma, the factor VIII level may, therefore, be derived from the clotting time of the second stage.

Chromogenic FVIII Assay

The assay is similar to the two-stage FVIII assay shown above in that it involves an incubation step to generate FXa and a second stage to determine the amount of FXa produced. In this case the amount of FXa is measured by its action on a highly specific chromogenic substrate and since the colour intensity produced is directly proportional to the amount of FXa, which in turn is directly proportional to the amount of FVIII, the FVIII levels may be calculated from the absorbance of the sample at a specific wavelength (the optimal absorbance wavelength for the chromophore produced by FXa cleavage of the chromogen, e.g. 405nm for the commonly used S-2765 chromogen).

A chromogenic FVIII assay requires:

Component Explanation
Reagent cocktail for generating FXa Contains FIXa, FX in excess, thrombin, a source of calcium ions and phospholipid
Chromogenic substrate A substance cleaved by FXa to produce a colour change. May also contain a thrombin inhibitor to stop the FXa generation when the chromogen is added
Patient plasma Platelet poor plasma

The patient’s plasma is incubated with the reagent cocktail at 37°C. The thrombin in the cocktail activates the FVIII to FVIIIa and, in the presence of Ca2+ and phospholipid, this acts as a co-factor for FIXa for the conversion of FX to FXa. FVIII is the rate limiting step. The chromogenic substrate is added. After a second incubation period the absorbance at a specific wavelength is measured and compared to a reference curve to give the FVIII level.

A reference curve is drawn by plotting the absorbance at 405nm against concentration on linear graph paper as shown below. For example - if in the case of an unknown plasma sample the absorbance at 405nm was 0.2 - then from the graph below the concentration of FVIII:C is 56 IU/dl.

The data used to construct the graph is shown in the table below:

Concentration [IU/dL] Absorbance [405nm]
140 0.44
100 0.32
50 0.19
0 0.04

Interpretation

See Comments for the 1-stage/2-stage FVIII discrepancies.

Reference Ranges

Reference ranges may be expressed as either a percentage or in IU/dl
The factor VIII reference range is usually 50-150% alternatively expressed as 0.50-1.50 IU/ml or 50-150 IU/dL. The reference range for neonates is very similar to that of adults.

Data Interpretation

Click HERE to go to the Data Interpretation Exercises.