From: Elizabeth M. Van Cott, M.D., and Michael Laposata, M.D., Ph.D., "Coagulation." In: Jacobs DS et al, ed. The Laboratory Test Handbook, 5th Edition. Lexi-Comp, Cleveland, 2001; 327-358.

Index of Tests

Activated Protein C Resistance and the Factor V Leiden Mutation [CO004550]

Related Information

Synonyms Activated Protein C Resistance; APC; Protein C Resistance, Activated

Applies to Factor V Leiden Mutation

Abstract Resistance to activated protein C (APC) is a condition which leads to a hypercoagulable state with an increased risk for venous thrombosis. The effect of exogenous APC on patient's clotting time [usually activated partial thromboplastin time (PTT)] is used to detect presence of resistance to APC (as occurs in individuals with the factor V Leiden mutation). A few laboratories might use clotting times other than the PTT. DNA-based assays can be used to directly detect the presence of the factor V Leiden mutation.

Specimen Plasma (for clotting time-based screening assay) and whole blood (for DNA-based confirmatory assay)

Container Blue top (sodium citrate) tube for screening assay. Container varies with laboratory for DNA-based assay (blue top, yellow top, or lavender top).

Collection Routine venipuncture. If multiple tests are being drawn, draw blue top tubes after any red top tubes but before any lavender top (EDTA), green top (heparin), or gray top (oxalate/fluoride) tubes. Immediately invert tube gently at least 4 times to mix. Tubes must be appropriately filled. Deliver tubes immediately to the laboratory.

Storage Instructions Clotting-time based assay: Store plasma at 4degrees C or room temperature if testing is performed within 4 hours; otherwise, store frozen until testing. According to one manufacturer, storage up to 24 hours without freezing is acceptable (Coatest APC Resistance V by Chromogenix). DNA-based assay: Store whole blood at 4degrees C or at room temperature.

Causes for Rejection Tube not full, specimen clotted, specimen received more than 4-24 hours after collection

Turnaround Time Clotting-time based assay: usually less than 1 day. DNA-based assay: several days (depending on how often test batches are performed).

Special Instructions Do not centrifuge or freeze whole blood specimen for DNA-based assay.

Reference Interval APC prolongs the PTT usually more than twofold in controls (normal persons) and less than twofold in affected individuals.

Some laboratories report the result as a normalized ratio, which is the result of the activated protein C resistance assay for the patient, divided by the result for normal pooled plasma.

Use The clotting-time assay identifies individuals who have resistance to APC. The DNA test identifies factor V Leiden as the cause of the APC resistance. Activated protein C resistance is the most prevalent hereditary predisposition to venous thrombosis. It is present in 5% of the general Caucasian population and is less common or rare in other ethnic groups.1,2,3 It accounts for 20% of unselected patients with a first deep vein thrombosis and 50% of familial cases of thrombosis.4,5,6 The vast majority of cases are due to the factor V Leiden mutation, which renders factor V resistant to degradation by activated protein C, resulting in an increased risk for venous thrombosis.7,8

Limitations Lupus anticoagulants, hirudin, or argatroban may cause inaccurate results in the commonly used PTT clotting-time based assay but do not affect DNA-based tests. Various alternative assays are not affected by lupus anticoagulants.9,10,11,12

Methodology Clotting time (usually PTT)-based. Test provides a measure of the APC-dependent prolongation of the clotting time (PTT), in essence, of the ability of APC to act as an anticoagulant. The specimen is first diluted 1:5 in factor V deficient plasma. An activated partial thromboplastin time (PTT) is performed on the diluted specimen, in the presence and absence of exogenously supplied activated protein C.4,13 Exogenous activated protein C degrades the patient's factors Va and VIIIa, thereby prolonging the PTT. The ratio of the PTT with activated protein C divided by the baseline PTT is calculated. Normal individuals usually have a ratio >2.0, whereas individuals with factor V Leiden usually have a ratio <2.0 because their mutated factor Va resists activated protein C degradation (each laboratory determines its own reference range). If the result is abnormal, a DNA-based assay (eg, polymerase chain reaction (PCR)-based assay) should be performed to determine if the patient has the factor V Leiden mutation, which confers activated protein C resistance. DNA-based methods allow precise determination of heterozygosity and homozygosity for the mutation.

The sensitivity and specificity of the PTT-based assay for detection of factor V Leiden mutation approach 100%.14 Some laboratories do not include the dilution into factor V deficient plasma described above, in which case the sensitivity is reduced to 50% to 86% and the specificity is reduced to 75% to 98% for detecting the factor V Leiden mutation.15,16,17 In addition, patients with an abnormal baseline PTT (usually including patients receiving Coumadin® or heparin) cannot be tested without the dilution step. Whether or not the test without the dilution step provides information regarding hypercoagulability is currently under investigation.18

Additional Information The anticoagulant action of activated protein C normally involves degradation of activated factors V and VIII by proteolytic cleavage at specific arginine residues, thereby inhibiting coagulation. Individuals with factor V Leiden have a mutation at one of the arginine cleavage sites in factor V, such that factor V resists degradation by activated protein C. The factor V Leiden mutation is a point mutation in which the guanine at nucleotide position 1691 is replaced by an adenine, resulting in substitution of arginine with glutamine at amino acid residue 506. One, and possibly two, additional factor V mutations at another arginine cleavage site are a very rare cause of activated protein C resistance,19,20 and other factor V mutations are also under investigation.21 Mutations in the factor VIII gene causing resistance to activated protein C are theoretically possible but have not yet been described. Using the normalized ratio reduces intra- and interlaboratory variability in the assay. However, it has not improved the ability of the assay to distinguish activated protein C resistance from normal.8,22,23

See Hypercoagulation Panel.


1. Hooper WC, Dilley A, Ribeiro MJA, et al, "A Racial Difference in the Prevalence of the Arg506 (RIGHT ARROW) Gln Mutation,"Thromb Res, 1996, 81(5):577-81.

2. Rees DC, Cox M, and Clegg JB, "World Distribution of Factor V Leiden,"Lancet, 1995, 346(8983):1133-4.

3. Ridker PM, Miletich JP, Hennekens CH, et al, "Ethnic Distribution of Factor V Leiden in 4047 Men and Women,"J Am Med Assoc, 1997, 277(16):1305-7.

4. Svensson PJ and Dahlbäck B, "Resistance to Activated Protein C as a Basis for Venous Thrombosis,"N Engl J Med, 1994, 330(8):517-22.

5. Griffin JH, Evatt B, Wideman C, et al, "Anticoagulant Protein C Pathway Defective in Majority of Thrombophilic Patients,"Blood, 1993, 82(7):1989-93.

6. Koster T, Rosendaal FR, de Ronde H, et al, "Venous Thrombosis Due to Poor Anticoagulant Response to Activated Protein C: Leiden Thrombophilia Study,"Lancet, 1993, 342(8886-7):1503-6.

7. Bertina RM, Koeleman BPC, Koster T, et al, "Mutation in Blood Coagulation Factor V Associated With Resistance to Activated Protein C,"Nature, 1994, 369(6475):64-7.

8. Voelkerding KV, Wu L, Williams EC, et al, "Factor V R506Q Gene Mutation Analysis by PCR-RFLP: Optimization, Comparison With Functional Testing for Resistance to Activated Protein C, and Establishment of Cell Line Controls,"Am J Clin Pathol, 1996, 106(1):100-6.

9. Akhtar MS, Blair AJ, King TC, et al, "Whole Blood Screening Test for Factor V Leiden Using a Russell Viper Venom Time-Based Assay,"Am J Clin Pathol, 1998, 109(4):387-91.

10. Le DT, Griffin JH, Greengard JS, et al, "Use of a Generally Applicable Tissue-Factor-Dependent Factor V Assay to Detect Activated Protein C-Resistant Factor Va in Patients Receiving Warfarin and in Patients With a Lupus Anticoagulant,"Blood, 1995, 85(7):1704-11.

11. Martorell JR, Munoz-Castillo A, and Gil JL, "False-Positive Activated Protein C Resistance Test Due to Antiphospholipid Antibodies Is Corrected by Platelet Extract,"Thromb Haemost, 1995, 74(2):796-7.

12. van Oerle R, van Pampus L, Tans G, et al, "The Clinical Application of a New Specific Functional Assay to Detect the Factor V (Leiden) Mutation Associated With Activated Protein C Resistance,"Am J Clin Pathol, 1997, 107(5):521-6.

13. Dahlbäck B, Carlsson M, and Svensson PJ, "Familial Thrombophilia Due to a Previously Unrecognized Mechanism Characterized by Poor Anticoagulant Response to Activated Protein C: Prediction of a Cofactor to Activated Protein C,"Proc Natl Acad Sci U S A, 1993, 90(3):1004-8.

14. Jorquera JI, Montoro JM, Fernandez MA, et al, "Modified Test for Activated Protein C Resistance,"Lancet, 1994, 344:1162-3.

15. Strobl FJ, Hoffman S, Huber S, et al, "Activated Protein C Resistance Assay Performance: Improvement by Sample Dilution With Factor V-Deficient Plasma,"Arch Pathol Lab Med, 1998, 122(5):430-3.

16. Sweeney JD, Blair AJ, and King TC, "Comparison of an Activated Partial Thromboplastin Time With a Russell Viper Venom Time Test in Screening for Factor V (Leiden) (FVR506Q),"Am J Clin Pathol, 1997, 108(1) :74-7.

17. Zehnder JL and Benson RC, "Sensitivity and Specificity of the APC Resistance Assay in Detection of Individuals With Factor V Leiden,"Am J Clin Pathol, 1996, 106(1):107-11.

18. de Visser MC, Rosendaal FR, and Bertina RM, "A Reduced Sensitivity for Activated Protein C in the Absence of Factor V Leiden Increases the Risk of Venous Thrombosis,"Blood, 1999, 93(4):1271-6.

19. Chan WP, Lee CK, Kwong YL, et al, "A Novel Mutation of Arg 306 of Factor V Gene in Hong Kong Chinese,"Blood, 1998, 91(4):1135-9.

20. Williamson D, Brown K, Luddington R, et al, "Factor V Cambridge: A New Mutation (Arg306 (RIGHT ARROW) Thr) Associated With Resistance to Activated Protein C,"Blood, 1998, 91(4):1140-4.

21. Faioni EM, "Factor V HR2: An Ancient Haplotype Out of Africa - Reasons for Being Interested,"Thromb Haemost, 2000, 83(3):358-9.

22. Brandt G, Gruppo R, Gluek CJ, et al, "Sensitivity, Specificity, and Predictive Value of Modified Assays for Activated Protein C Resistance in Children,"Thromb Haemost, 1998, 79(3):567-70.

23. Tripodi A, Chantarangkul V, Negri B, et al, "Standardization of the APC Resistance Test: Effects of Normalization of Results by Means of Pooled Normal Plasma,"Thromb Haemost, 1998, 79(3):564-6.


Dahlbäck B, "Molecular Genetics of Thrombophilia: Factor V Gene Mutation Causing Resistance to Activated Protein C as a Basis of the Hypercoagulable State,"J Lab Clin Med, 1995, 125(5):566-71.

De Stefano V, Finazzi G, and Mannucci PM, "Inherited Thrombophilia: Pathogenesis, Clinical Syndromes, and Management,"Blood, 1996, 87(9):3531-44.


Comments/Questions - Report a Change - Disclaimer
This page last updated on March 17, 2010