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Screening test for the factor V Leiden gene mutation and other causes of activated protein C resistance
This assay detects thrombotic risk factors other than factor V Leiden. This test should not be performed on individuals with extended baseline aPTT values due to the presence of lupus anticoagulants.6 This test should not be used for patients receiving thrombin inhibitors such as hirudin and argatroban.6 In some cases, this test will fail to distinguish individuals who are heterozygous for the factor V Leiden mutation from normals.7 Genetic testing may be required to distinguish between heterozygous and homozygous factor V Leiden mutation.7 Elevated factor VIII levels, as can be seen in acute phase reaction, can normalize the aPTT and effectively reduce the anticoagulant effect of aPC.7 Abnormal results can also be seen in pregnancy, especially during the third trimester, due to decreased levels of protein S and increased levels of factors V and VIII.7
This assay measures the effect of adding aPC on the activated partial thromboplastin time (aPTT) of the patient sample.8,9 An aPTT is performed twice, once with and again without added aPC. The ratio of the two clotting times is reported. The patient sample is diluted with factor V-deficient plasma. This serves to correct for any factor deficiencies in the patient sample and allows the test to be used for patients on oral anticoagulants. The reaction mixture also contains a heparin neutralizer to allow this test to be performed on patients receiving heparin therapy.
Activated protein C (aPC), in a complex with protein S, inactivates procoagulant factors Va and VIIIa by proteolytic cleavage at specific arginine residues.7,10,11 This serves to control coagulation and limit the extent of thrombus formation. The functionality of the aPC inhibitory system in a given individual can be assessed through an in vitro clotting assay. Addition of aPC to a patient's plasma serves to extend the activated partial thromboplastin time (aPTT) for individuals who are sensitive to aPC. Individuals are considered to be aPC resistant when addition of aPC fails to extend the time to clot formation in this assay.
More than 95% of cases of aPC resistance are caused by a specific polymorphism in the factor V gene that is referred to as factor V Leiden.8 This single point mutation results in a substitution of glutamine for arginine at amino acid number 506 of factor V. Arginine number 506 is an aPC cleavage site of normal factor V, making factor V Leiden resistant to inactivation by aPC. Heterozygous factor V Leiden mutation occurs in 3% to 7% of persons of Northern European descent. However, this mutation is essentially absent in Asian, African, and Mediterranean populations.8 Individuals with heterozygous factor V Leiden have a three- to tenfold increased risk of thrombosis.8 The risk of thrombosis is approximately 80-fold increased in individuals who are homozygous for factor VLeiden.8 The incidence of factor V Leiden in unselected individuals experiencing their first venous thrombosis or pulmonary embolism is approximately 20%. In patients with a family history of thrombophilia, the likelihood of factor VLeiden being the cause reaches as high as 50%.8 Risk of thrombosis is further increased if the factor V Leiden mutation is accompanied by other prothrombotic conditions (ie, antithrombin, protein C or protein S deficiency, prothrombin G20210, pregnancy, oral contraceptive usage, prolonged immobilization, or surgery).
While factor V Leiden is, by far, the most common genetic cause of aPC resistance, other polymorphisms can produce this condition.9 aPC resistance can occur due to an elevated factor VIII level, in itself a risk factor for thrombosis.9 Factor V Cambridge, a very rare mutation at another arginine residue of factor V, can produce aPC resistance.7,8 The HR2 haplotype, also referred to as A4070G mutation, is another rarely observed genetic cause of aPC resistance.7
Citrated plasma samples should be collected by double centrifugation. Blood should be collected in a blue-top tube containing 3.2% buffered sodium citrate.1 Evacuated collection tubes must be filled to completion to ensure a proper blood to anticoagulant ratio.2,3 The sample should be mixed immediately by gentle inversion at least six times to ensure adequate mixing of the anticoagulant with the blood. A discard tube is not required prior to collection of coagulation samples, except when using a winged blood collection device (ie, "butterfly"), in which case a discard tube should be used.4,5 When noncitrate tubes are collected for other tests, collect sterile and nonadditive (red-top) tubes prior to citrate (blue-top) tubes. Any tube containing an alternate anticoagulant should be collected after the blue-top tube. Gel-barrier tubes and serum tubes with clot initiators should also be collected after the citrate tubes. Centrifuge for 10 minutes and carefully remove 2/3 of the plasma using a plastic transfer pipette, being careful not to disturb the cells. Deliver to a plastic transport tube, cap, and recentrifuge for 10 minutes. Use a second plastic pipette to remove the plasma, staying clear of the platelets at the bottom of the tube. Transfer the plasma into a LabCorp PP transpak frozen purple tube with screw cap (LabCorp N° 49482). Freeze immediately and maintain frozen until tested. To avoid delays in turnaround time when requesting multiple tests on frozen samples, please submit separate frozen specimens for each test requested.
Please print and use the Volume Guide for Coagulation Testing to ensure proper draw volume.
Causes for Rejection
If the patient's hematocrit exceeds 55%, the volume of citrate in the collection tube must be adjusted. Refer to Coagulation Collection Procedures for directions.
|Order Code||Order Code Name||Order Loinc||Result Code||Result Code Name||UofM||Result LOINC|
|117762||Activated Protein C Resistance||13590-5||117763||Act.Prt.C Resist.||ratio||13590-5|
|Reflex Table for Act.Prt.C Resist.|
|Order Code||Order Name||Result Code||Result Name||UofM||Result LOINC|