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