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A Practical Guide to Laboratory Haemostasis

 

Resistance to Activated Protein C [APCr Assays]



Introduction

Resistance to Activated Protein C [APCr] was first reported in 1995 and in approximately 95% of cases is due to the Factor V Leiden [FVL] mutation – a G1691A missense mutation at Arginine 506 resulting in its replacement by a glutamine [R506Q] and the abolition of an APC inactivation cleavage site in Factor Va. This is illustrated diagrammatically below [T - thrombin cleavage site; APC - activated Protein C.]

Principles & Method

A variety of tests have been developed to screen for APC resistance:

Method Explanation
APTT The original test for APC resistance involved measuring the APTT of a plasma sample with or without the addition of exogenous APC. In a plasma sample without APCr, the addition of APC inactivates factor Va and factor VIIIa and so prolongs the clotting time of the APTT. In contrast in a sample with the Factor V Leiden mutation, the prolongation in the clotting time is less.

A ratio is derived from: [APTT+APC]/[APTT-APC]

A imitation of this test is that it requires a normal APTT in the patient and so cannot be used in cases in which there is a prolongation of the APTT e.g. patients on oral anticoagulants or with a lupus anticoagulant.

Individuals without the FVL mutation generally have a ratio of >2.0 and individuals who are heterozygous for the FVL mutation have a ratio <2. However, their is considerable overlap between healthy individuals and heterozygotes.
Normalised APTT The normalised APTT was established to try and improve the discrimination between FVL heterozygotes and individuals without the FVL mutation. The normalised ratio is derived by first dividing the APTT in the presence of APC by the APTT in the absence of APC [as with the original APTT screening test above.] This ratio is then normalized against a reference pool to obtain an APC sensitivity ratio.

[APTT+APC]/[APTT-APC] of patient sample
[APTT+APC]/[APTT-APC] of a normal reference plasma pool

80% of patients with an APC sensitivity ratio <0.84 and 100% of patients with an APC sensitivity ratio <0.70 were heterozygous or homozygous respectively for the FVL mutation.

It is important to remember that the presence in the normal plasma pool of a mutant FV [i.e. someone who is heterozygous for the FVL mutation] may reduce the APC ratio of the normal plasma pool.
Modified APTT with Pre-dilution in FV-deficient plasma
Pefakit® APC-R Factor V Leiden screen The PefakitŪ APC-R Factor V Leiden screen is a plasma based functional clotting assay and differs from other functional APC resistance tests by using a specific FV activator isolated from a snake venom (RVV-V from Daboia russelli).
The test is performed with and without the addition of APC and involves a pre-dilution [1 +4] of patient plasma with factor V-deficient plasma.

Coagulation is triggered by the addition of a FV dependent prothrombin activator isolated from the venom [Noscarin] another snake [Notechis scutatus scutatus] in the absence of calcium. The time for clot formation is recorded and the ratio:
(Clotting Time + APC/Clotting Time - APC] determined. 

If the FVa molecules have been eliminated during the incubation step, the velocity of prothrombin activation by the FV-dependent Noscarin is slow and therefore, the clotting tim is prolonged.

The reagents employed in this test contain polybrene which makes it insensitive to heparin up to a concentration of <2 IU/ml (UFH and LMWH) or pentasaccharide levels <2 µg/ml.
Chromogenic Assay The chromogenic assay for detecting APC resistance is based on the capacity of APC to limit the generation of factor Xa by inactivating factor Vllla in plasma. The ratio of the factor Xa amidolytic activity in a sample without APC to its factor Xa activity with the addition of APC reflects the response of the plasma coagulation system to APC. 
Russell Viper Venom Assay This test is based on the dilute Russell Viper Venom Time (DRVVT). The DRVVT is prolonged when a plasma sample is pre-incubated with a diluted snake venom isolated from Agkistrodon contortrix contortrix which activated Protein C.
The test result is expressed as the ratio between the DRVVT with and without addition of the venom.  

Interpretation

The interpretation of the test is very much dependent upon the specific test. Variables that have been shown to affect the test include:
↑ APTT e.g. anticoagulants, Lupus anticoagulant
↑ FVIII e.g. pregnancy

Reference Ranges

These very much depend upon the specific test. A test ideally should be able to differentiate normal individuals without APCr from individuals who are heterozygous or homozygous for the FVL mutation. However - see Comment 1 above.

What Test Next

Individuals with a low APC ratio usually go on to have a screen of their F5 gene for the Factor V Leiden mutation. However, it should be remembered that although most cases of APC resistance are due to the Factor V Leiden mutation, testing with the original APTT-based APC resistance assay may be useful in detecting independent risk factors for venous thrombosis including pregnancy, oral contraceptive use, and the presence of a lupus anticoagulant. APC resistance independent of the FV Leiden mutation appear to a risk factor for VTE. Some F5 mutations associated with APCr but which are not due to the FVL Arg506Gln mutation have also been reported e.g. FV Cambridge.

Data Interpretation

Click HERE to go to the Data Interpretation Exercises.