Purpose of the test
To identify in a patient a possible predisposition to thrombosis. The defect is usually inherited but may be acquired in some cases. May be extremely useful to prevent DVT/PE in patients with high risk factors such as long haul flight.
Antithrombin III is a naturally occurring inhibitor. It will inactivate serine proteases and has a special affinity with thrombin and Factor Xa.
In this functional assay plasma is incubated with an excess of Factor Xa in the presence of heparin, which is a co-factor of AT III. The complex AT III:heparin binds rapidly to Xa. The amount of Xa bound is determined by the concentration of AT III in sample. The residual free Factor Xa is measured by its cleavage of a chromogenic substrate. The para-nitroaniline dye released gives a yellow colour which is inversely proportional to AT III concentration.
Protein C activity
Protein C is a vitamin K dependent protein synthesised in the liver. It is activated by thrombin in the presence of calcium ions and phospholipid and the process is accelerated by thrombomodulin.
Activated Protein C Resistance
Activated Protein C is enhanced by a co-factor, Protein S, and regulates the coagulation process by neutralising Factors Va and VIIIa.
Protein C is activated by Protac, a specific enzyme derived from the venom of the Copperhead snake (Agkistrodon c. contortrix). Activated Protein C cleaves the specific chromogenic substrate and the para-nitroaniline dye released is measured at 405 nm.
Activated Protein C Resistance (APCR) is a hereditary defect of the Protein C anticoagulant pathway which results in a pre-disposition to thrombo-embolic disease. Activated Protein C, together with its cofactor, Protein S, is an important down-regulator of thrombin formation by inhibiting the activity of activated Factor V (Va) and Factor VIII (VIIIa) in vivo. The phenomenon can be demonstrated in vitro by an abnormally reduced anti-coagulant response in human plasma upon the addition of human Activated Protein C (APC). Therefore, in the classic test the APTT is performed with and without the addition of APC. The basal APTT will be significantly prolonged by the addition of APC in normal individuals due to the deactivation of Factors Va and VIIIa leading to a reduced rate of thrombin formation. Individuals with APCR have significantly shorter APTTs upon the addition APC compared with normals. The result is expressed as a ratio of clotting time with APC divided by basal clotting time without APC. The APC Ratio will be higher in normal individuals. Inherited resistance to activated protein C, associated with the factor V Leiden mutation G1691A has been shown to be present in 20-50% of individuals with a history of deep vein thrombosis (DVT). G to A transition at position 20210 in the 3’ untranslated region of the prothrombin gene has also been implicated in the pathogenesis of venous thrombosis with an incidence of 5-7% in patients with at least one confirmed episode of DVT.
Heterozygosity for the Prothrombin G20210A variant has been shown to increase the risk of venous thrombosis by up to 3 fold. (Prothrombin G20210A genotype G/A)
Heterozygosity for factor V Leiden mutation is associated with a 5-10 fold increased risk of thrombosis. (Factor V Leiden genotype G/A)
The presence of Homozygous Factor V Leiden (Factor V Leiden genotype A/A) has been shown to increase the risk of venous thrombosis by 50-100 fold.
Lupus anticoagulants (LA) are also known as anti-phospholipid antibodies. Their presence is known to cause arterial and venous thrombosis, recurrent spontaneous abortions, thrombocytopaenia and neurological problems. They are found in association with auto-immune conditions, exposure to some drugs, some infections, and lymphoproliferative disorders. The antibodies are a heterogeneous group which interfere with the action of anionic phospholipids. This interference can be demonstrated by prolongation of clotting times in phospholipid-dependent clotting tests, such as the Activated Partial Thromboplastin Time (APTT) and the dilute Russell’s Viper Venom test (DRVV)
ANALYTE: Antithrombin III
REFERENCE RANGE u / dl : 80 - 130
ANALYTE:Protein C activity
REFERENCE RANGE u / dl : 70 - 140
ANALYTE:APCRV (with pre-dilution in Factor V deficient plasma) REFERENCE RANGE u / dl : 2.0 – 3.0
Lupus anticoagulant screen:
DVVTest ratio <1.15 is negative for Lupus Anticoagulant.
DVVTest ratio >1.20 AND
% correction of ratio >12% = positive for Lupus Anticoagulant.
If a patient is normal for FVL:
Factor V Leiden Genotype: G/G = NORMAL
3 x 4.5 mls tri-sodium citrate samples and 1 EDTA sample.
Storage and Transport
Sample should be send directly to the laboratory and must be processed or prepared for storage within 2 hours of venipuncture.
Samples should be transported to the laboratory at room temperature.
If frozen, the plasma must be transported on dry ice to preserve the –40°C temperature
Batches of tests are run weekly.
Factors affecting results or interpretation
Clots of any size , haemolysis, underfilling or overfilling will affect result.
Presence of heparin anticoagulant will inhibit PCR applications.
Clotted samples are unsuitable for DNA analysis.
Samples must be clearly labelled with the patients first name, surname, D.O.B, hospital number and the date the sample was taken. The details on the sample must correspond to the request form. Unlabelled samples will not be accepted.
Haemostasis Laboratory on 020 3299 9000 ext 2434