Venous thromboembolism (VTE) consists of deep vein

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1 Clinical Utility of Factor V Leiden (R506Q) Testing for the Diagnosis and Management of Thromboembolic Disorders Richard D. Press, MD, PhD; Kenneth A. Bauer, MD; Jody L. Kujovich, MD; John A. Heit, MD Objective. To review the current state of the art regarding the role of the clinical laboratory in diagnostic testing for the factor V Leiden (FVL) thrombophilic mutation (and other protein C resistance disorders), and to generate, through literature reviews and opinions of recognized thought-leaders, expert consensus recommendations on methodology and diagnostic, prognostic, and management issues pertaining to clinical FVL testing. Data Sources, Extraction, and Synthesis. An initial thorough review of the medical literature and of current best clinical practices by a panel of 4 experts followed by a consensus conference review, editing, and ultimate approval by the majority of a panel of 28 additional coagulation laboratory experts. Conclusions. Consensus recommendations were generated for topics of direct clinical relevance, including (1) defining those patients (and family members) who should (and should not) be tested for FVL; (2) defining the preferred FVL laboratory testing methods; and (3) defining the therapeutic, prophylactic, and management ramifications of FVL testing in affected individuals and their family members. As FVL is currently the most common recognized familial thrombophilia, it is hoped that these recommendations will assist laboratorians and clinicians caring for patients (and families) with this common mutation. (Arch Pathol Lab Med. 2002;126: ) Venous thromboembolism (VTE) consists of deep vein thrombosis (DVT) and its complication, pulmonary embolism. Most pulmonary emboli arise from the proximal deep veins of the leg (popliteal, superficial femoral, or common femoral veins) or pelvis (iliac veins), but they also may arise from isolated deep veins of the calf or from axillary/subclavian veins. Deep vein thrombosis also may affect the caval, innominate, cerebral, hepatic, portal, splenic, mesenteric, and renal venous circulations. Venous thromboembolism is the third most common cardiovascular disease in the United States, with an incidence of approximately 1 per 1000 person-years (0.1%) and a lifetime clinical prevalence of about 5%; VTEs account for deaths annually. 1 3 Venous thromboembolism is a common complex (multifactorial) disease, exhibiting genetic locus heterogeneity with incomplete penetrance and variable expressivity, as well as genetic and environmental interactions. Mutations in several different genes associate with VTE, although not Accepted for publication June 21, From the Departments of Pathology and Medical Genetics (Dr Press) and Medicine (Dr Kujovich), Oregon Health & Science University, Portland; the Department of Medicine, Veterans Affairs Boston Healthcare System and Beth Israel Deaconess Medical Center, West Roxbury, Mass (Dr Bauer); and the Division of Cardiovascular Diseases, Mayo Clinic, Rochester, Minn (Dr Heit). Presented at the College of American Pathologists Consensus Conference XXXVI: Diagnostic Issues in Thrombophilia, Atlanta, Ga, November 9 11, Reprints: Richard D. Press, MD, PhD, Department of Pathology L113, Oregon Health & Science University, 3181 SW Sam Jackson Park Rd, Portland, OR ( pressr@ohsu.edu). all mutation carriers develop VTE (incomplete penetrance), and the severity and age of onset of VTE may vary (variable expressivity). Simultaneous mutations within 2 or more genes, or homozygosity for mutations at 1 locus, compound the VTE risk (genetic interaction). Among mutation carriers, exposure to clinical (environmental) risk factors also compounds the risk of VTE (environmental interaction). Independent risk factors for VTE include older age (continuous risk), male sex, confinement to a hospital or nursing home, recent surgery that required an anesthesia, trauma sufficient to require hospitalization, malignant neoplasm (with or without chemotherapy), neurologic disease with chronic extremity paresis, superficial vein thrombosis, and prior central venous catheter or transvenous pacemaker (for upper extremity DVT). 3 Additional VTE risk factors for women include the use of oral contraceptives, estrogen replacement therapy, tamoxifen, and raloxifene. 4,5 Serious liver disease is associated with a 90% decrease in risk for VTE. 3 Venous thromboembolism also recurs frequently. The estimated cumulative incidence of first VTE recurrence is 1.6% at 7 days, 5.2% at 30 days, 8.3% at 90 days, 10.1% at 180 days, 12.9% at 1 year, 16.6% at 2 years, 22.8% at 5 years, and 30.4% at 10 years. 3 The hazard rate per 1000 person-days ( SD) for recurrence is highest in the first 6 to 12 months after the initial event, ranging from recurrent VTE events at 7 days, to events at 30 days, 30 5 events at 90 days, 20 4 events at 180 days, and 20 2 events at 1 year. However, the recurrence hazard rate never falls to zero, continuing at 10 1 events at 2 years, 6 1 at 5 years, and 5 1 at 10 years. These data suggest that VTE is a chronic disease with episodic 1304 Arch Pathol Lab Med Vol 126, November 2002 Factor V Leiden Press et al

2 Table 1. Thrombophilic Defect Incident VTE Recurrent VTE Antithrombin deficiency Protein C deficiency Protein S deficiency APC resistance/factor V Leiden (R506Q) Prothrombin G20210A mutation * VTE indicates venous thromboembolism; APC, activated protein C. Genetic Alterations in Thrombophilia* Population Prevalences, % (Whites) Normal Population Relative Thrombotic Risk (heterozygotes) (homozygotes) Genetic Alterations Heterogeneous Heterogeneous Heterogeneous Single nucleotide polymorphism (heterozygotes) Single nucleotide polymorphism recurrence. Independent predictors of recurrence include older age, obesity, malignant neoplasm, and extremity paresis. 6 Thrombophilia (eg, hypercoagulable or prothrombotic disorder) is defined as an acquired or inherited predisposition to thrombosis. The phenotypic ( clinical ) presentations of thrombophilia may include VTE, recurrent miscarriage, complications of pregnancy (preeclampsia, abruptio placentae, intrauterine growth retardation, stillbirth), and possibly stroke, acute coronary syndromes (unstable angina, non Q-wave and Q-wave myocardial infarction), and aseptic necrosis of the femoral head. A clinical suspicion of an underlying thrombophilia should be considered for patients with VTE onset at a younger age (before the age of 50 years), recurrent thrombotic events, a family history of VTE, VTE at unusual anatomic sites (cerebral, mesenteric, portal, or hepatic veins, or the vena cava), multiple adverse pregnancy outcomes, or unprovoked idiopathic VTE. The majority of patients carrying the most common mutation associated with thrombophilia, factor V Leiden (FVL), suffer DVT of the lower extremity at an older age and at the time of exposure to wellrecognized risk factors (eg, surgery, trauma, oral contraceptives, or estrogen replacement therapy). Approximately 50% of all VTE patients without malignancy will have a detectable acquired or familial thrombophilia. Up to 70% of typical thrombophilic patients (as defined above) will be found to have 1 or several of the 5 major inherited defects listed in Table 1. Two of these genetic defects (FVL and prothrombin G20210A), which together are found in more than half of all cases of inherited thrombophilia, are newly described (since 1994), well-conserved single nucleotide substitutions for which direct DNA-based assays are available. In contrast, the more widely recognized familial deficiencies in the anticoagulant proteins antithrombin, protein C, and protein S together are found in less than 10% of all VTE patients. However, historically, these deficiencies have been more frequent targets for clinical thrombophilia investigations than the much more common FVL mutation. 7 This inequity in appropriate laboratory testing practice suggests that laboratory professionals should become more active in providing expert laboratory practice consultation to the generalist physicians treating the majority of these patients. Toward that end, the College of American Pathologists convened a panel of thrombophilia experts to develop a set of consensus recommendations on clinical FVL testing based on published scientific evidence. The criteria used to categorize these evidence levels are shown in Table 2. The expert panel s consensus recommendations Table 2. Levels of Evidence for Consensus Recommendations Level 1: The recommendation is based on 1 or more welldesigned prospective studies or 2 or more welldesigned retrospective studies Level 2: The recommendation is based on retrospective studies or multiple anecdotal studies that reach consensus Level 3: The recommendation is based on isolated anecdotal studies and/or the consensus of expert practitioners (and corresponding levels of evidence) are presented in Tables 3 and 4. FACTOR V R506Q (LEIDEN) AND ACTIVATED PROTEIN C RESISTANCE Before 1993, the evaluation of inherited thrombophilia was limited to plasma-based functional assays for protein C, protein S, or antithrombin deficiency, which together are found in less than 10% of patients with incident VTE. 8 The laboratory approach to thrombophilia testing changed in 1993, when Dahlback and colleagues 9 described a new and very common familial thrombophilia, hereditary resistance to activated protein C, which is identified by measuring the ratio of the activated partial thromboplastin time (aptt) clotting time, with and without the addition of exogenous activated protein C (APC). Other clinically affected relatives of the probands demonstrated APC resistance in the aptt-based test, suggesting that the abnormality was inherited. Several laboratories subsequently reported the specific genetic defect responsible for APC resistance a single well-conserved G to A missense mutation at nucleotide 1691 of the factor V gene The resulting amino acid substitution, namely, arginine (R) to glutamine (Q) at amino acid 506, occurs precisely at 1 of the 3 sites where APC normally cleaves and inactivates procoagulant factor Va. 14 Because of this single amino acid substitution, activated FVL is partially resistant to the anticoagulant action of APC and is inactivated at an approximately l0-fold slower rate than normal, resulting in increased thrombin generation and a prothrombotic state. In contrast to the genetic heterogeneity in patients with protein C, S, or antithrombin deficiency, approximately 90% to 95% of those with functional APC resistance, as measured by the clotting time test, have the identical factor V R506Q (Leiden) missense mutation. In rare cases, genetic abnormalities other than the factor V R506Q mutation produce the APC resistance phenotype or modulate its expression in factor V R506Q heterozygotes. 15,16 Several Arch Pathol Lab Med Vol 126, November 2002 Factor V Leiden Press et al 1305

3 Table 3. Consensus Recommendations for Factor V Leiden (FVL) Testing: Who Should Be Tested?* Conclusions 1. There is currently no evidence that the acute therapeutic management of venous thromboembolic events (duration and intensity of anticoagulation) should be different in patients with FVL. Hypothesis 2. The primary advantages of FVL testing would be the identification of high-risk patients who could benefit from either: long-term anticoagulant therapy, or aggressive prophylaxis in temporary periods of high thrombotic risk. 3. Other direct clinical benefits of FVL testing would include the opportunity to detect: female probands for whom future decisions as to oral contraceptive use, hormone replacement therapy, or management of pregnancy complications could depend on FVL carrier status, or at-risk family members for whom future decisions as to antithrombotic prophylaxis, oral contraceptive use, hormone replacement therapy, or pregnancy complications could depend on FVL carrier status. Recommendations for FVL testing As the discovery of an FVL mutation (by itself or in combination with other thrombophilias) would, in some situations, directly alter clinical management of the proband or lead to testing of family members, FVL testing is recommended in patient populations with a mutation prevalence above that of the normal population, such as those with venous thromboembolism and a clinical suspicion of thrombophilia based on any of the following criteria. 4. FVL testing is recommended in patients with: a history of recurrent VTE, Level 2 42,44,192 a first VTE at younger than 50 years, Level 1 18,19,39 a first unprovoked VTE at any age, Level 1 21,46 a first VTE at an unusual anatomic site, such as the cerebral, mesenteric, portal, or hepatic veins, Level a first VTE, at any age, in a subject with a first degree family member with a VTE before the age of 50 years, Level 1 170,171 a first VTE related to pregnancy, the puerperium, or oral contraceptive use, Level a first VTE related to hormone replacement therapy, Level 3 119,120,124,125 women with unexplained pregnancy loss during the second or third trimester. Level 2 35,37,137, ,157, Testing for FVL is controversial in: young women smokers (age 50 y) with a myocardial infarction, Level 2 93 older patients (age 50 y) with a first provoked VTE event in the absence of cancer or an intravascular device, Level 3 18,19,39 a first VTE related to SERMs or tamoxifen, Level 3 119,120,124,125 selected cases of women with unexplained severe preeclampsia, placental abruption, or intrauterine growth retardation. Level 3 137,148, After appropriate counseling, testing for FVL also may be indicated in: asymptomatic adult family members of probands with known FVL mutations, especially those with a strong family history of thrombosis at a young age ( 50 y), Level 2 17,170,171,177,197 asymptomatic female family members who are pregnant or are considering oral contraceptives or pregnancy. Level 2 135,170,171,197 Table 3. Continued 7. Factor V Leiden testing is not recommended: as a general population screen, Level 1 40,176 as a routine initial test during pregnancy, Level 2 115,130,135,136 as a routine initial test prior to or during oral contraceptive use (Level 2), hormone replacement therapy (Level 3), or SERM therapy (Level 3), 115,116 as a prenatal test, newborn initial test, or as a routine test in asymptomatic prepubescent children, Level 2 as a routine initial test in patients with arterial thrombotic events (Level 1). However, FVL testing can be considered in certain unusual situations, such as in patients with unexplained arterial thrombosis without atherosclerosis or in young patients who smoke. 19,85 91,93 * VTE indicates venous thromboembolism; SERMs, selective estrogen receptor modulators. Table 4. Consensus Recommendations for Factor V Leiden (FVL) Testing: FVL Testing Method Conclusions 1. The variable sensitivity and specificity of the first generation APC resistance assay precludes its routine clinical use in the evaluation of thrombophilia. However, the second generation APC resistance assay (with dilution of test plasma into factor V deficient plasma) has, in some laboratories, a diagnostic specificity approximately equivalent to direct DNA-based mutation tests and is currently less costly. 2. Definitive direct DNA-based methods to detect the FVL mutation are available in clinical diagnostic laboratories by any of several different methods developed in-house (non-fda approved). These direct mutation assays, when appropriately validated in a licensed clinical laboratory, are extremely accurate and precise for the detection of FVL. Recommendations for FVL testing method 3. For patients, initial FVL testing may include either the second generation APC resistance functional assay or a direct DNA-based mutation method. Level 2 24,26,27,30 Exceptions are noted as follows. 4. An initial DNA-based testing method is recommended in: patients with a lupus anticoagulant and a markedly prolonged baseline aptt (which may interfere with the functional APC resistance assay), Level 2 25 family members of subjects with known FVL mutations, in order to avoid the need for follow-up confirmatory direct mutation testing. Level 3 5. A negative second generation functional assay excludes the diagnosis of FVL. Confirmatory direct DNA-based testing is, however, recommended for patients with: borderline APCR values, Level 1 positive APCR initial tests to definitively confirm both the diagnosis and the number of mutant alleles, and, for patients with very low APCR values, to distinguish heterozygotes, homozygotes, and those who are heterozygous for both FVL and a second mutation causing factor V deficiency. Level 3 6. Laboratory testing for other inherited and acquired thrombophilic defects should be considered even after the identification of FVL, since the FVL allele often coexists with other disorders and, when present, synergistically increases the thrombotic risk. Level 1 21,62 65,67,68,70,173,198 * APC indicates activated protein C; FDA, Food and Drug Administration; aptt, activated partial thromboplastin time; and APCR, activated protein C resistance Arch Pathol Lab Med Vol 126, November 2002 Factor V Leiden Press et al

4 patients have been described with APC resistance of unknown cause. 17 Factor V Leiden is the most common inherited cause of thrombophilia, being present in heterozygous form in approximately 12% to 20% of incident VTE patients 18,19 and approximately 40% to 50% of those with recurrent or familial VTE. 20,21 About 3% to 7% of the normal white populations of northern European or Scandinavian ancestry are heterozygous FVL carriers. The FVL mutation is much less common in populations of non-european ancestry, with a carrier frequency of 1.2% in African Americans, 2.2% in Hispanic Americans, 1.2% in Native Americans, and 0.45% in Asian Americans. 22 Homozygotes account for about 1% of the white population with the FVL mutation, but are disproportionately overrepresented clinically because of their higher thrombotic risk. FUNCTIONAL TESTS FOR FACTOR V LEIDEN Since the FVL mutation creates a coagulation factor V that is 10 times less susceptible to APC-induced inactivation, functional assays for this alteration involve various methods for testing the ability of exogenously added APC to affect functional coagulation kinetics. The original APC resistance assay, now largely supplanted by superior methods, measured the ratio of aptt clotting times in the presence and absence of a standard amount of exogenous APC. 23 This first generation assay is based on the principle that, when added to normal plasma, APC inactivates factor Va (and possibly factor VIIIa), which slows coagulation and prolongs the aptt. The APC-resistant phenotype is characterized by a minimal prolongation of the aptt in response to APC and a correspondingly low ratio. Although the first generation assay is, in some laboratories, highly sensitive for the APC resistance phenotype, it has a variably poor specificity for the FVL mutation and cannot accurately distinguish heterozygotes from homozygotes. 24 Other limitations are that it cannot be used in patients with a prolonged baseline aptt due to warfarin or heparin anticoagulation, other coagulation defects, or a lupus inhibitor, and the results may be altered by the hemostatic changes during pregnancy, oral contraceptive use, or acute thrombosis. 25 The high prevalence of these confounders in patients being evaluated for thrombophilia limits the practical utility of this first generation functional assay. A modified (second generation) APC resistance functional assay that overcomes the limitations of the original method is now widely available (with Food and Drug Administration approval). In this assay, the patient s plasma is first diluted in factor V deficient plasma that contains a heparin neutralizer. The addition of the factor V deficient plasma corrects for deficiencies of other coagulation proteins, neutralizes therapeutic concentrations of heparin, and eliminates the effect of some lupus inhibitors. 26 The modified assay has a very high sensitivity and specificity for FVL 27 ; can accurately distinguish heterozygotes from homozygotes 28 ; and can be accurately interpreted in patients receiving heparin or warfarin, in many patients with lupus inhibitors, and in the setting of acute thrombosis, pregnancy, or inflammation. However, it will not identify the rare patient with APC resistance not due to factor V abnormalities, 29 and each individual laboratory needs to determine its own normal reference ranges. DIRECT MUTATION TESTS FOR FVL The factor V mutation responsible for APC resistance is a conserved single point mutation (G to A) at nucleotide 1691 in exon 10 of the factor V gene. Direct DNA-based methods to detect this mutation are increasingly available in clinical diagnostic laboratories as the definitive test for this disorder. Because Food and Drug Administration approved reagents are not yet commercially available, accurate and precise tests that have been developed in house are widely used. Commonly used molecular methods to detect the FVL mutation often involve multistep processes, beginning with a polymerase chain reaction (PCR) amplification of the region surrounding the exon 10 alteration. The amplification product is then analyzed by one of a number of allele-discriminatory methods, including allelespecific restriction enzyme digestion (PCR restriction fragment length polymorphism, in which the mutant allele is differentially recognized by a sequence-specific restriction endonuclease), allele-specific PCR amplification (in which the DNA template is either amplified or not amplified based on sequence-specific binding of an allele-specific PCR primer), or allele-specific hybridization (in which the PCR product is differentially hybridized to labeled allele-specific probes on solid supports). These multistep, labor-intensive, manual direct mutation assay formats are extremely accurate and precise. 30 However, because of their high cost and lack of automation, many laboratories now perform FVL genotyping by recently developed semiautomated, homogeneous, 1-step direct mutation methods. These faster, less laborious genotyping methods include fluorescent detection of real-time PCR products with allele-specific hybridization probes, 31 non-pcr signal amplification methods based on either enzymatic hybridization mismatch recognition using fluorescent allele-specific probes 32 or linked fluorescent allele-specific pyrophosphorolysis-kinase reaction, 33,34 and various other PCR and non-pcr based methods. These new genotyping methods, together with other advances in the automation of the DNA preparation, amplification setup, and amplicon product detection steps, promise to significantly reduce DNA-based test costs. In addition, since the clinical indications for direct DNA-based assays of other heritable thrombophilias (eg, prothrombin G20210A) are similar to those for FVL, novel methods to simultaneously evaluate multiple prothrombotic mutations will soon become clinically and economically advantageous. Semiautomated methods for these multitarget DNA assays under development include single-tube, multiprimer multiplex PCR formats, and target and allele-specific hybridizations on the surface of miniaturized solid supports (ie, DNA chips). Using these multigene platforms, novel prothrombotic target gene mutations identified by the completed human genome project could be added to existing thrombophilia gene panels. The future evaluation of thrombophilia may then ultimately include an individualized comprehensive risk assessment based on the sequence patterns of multiple relevant prothrombotic target genes. Recommendations for the optimal testing method for FVL detection are summarized in Table 4. FACTOR V LEIDEN AND VENOUS THROMBOSIS Factor V Leiden is the most common cause of hereditary thrombophilia. The major clinical manifestation is DVT with or without pulmonary embolism. The risk is com- Arch Pathol Lab Med Vol 126, November 2002 Factor V Leiden Press et al 1307

5 parably increased for cerebral vein thrombosis. There is also evidence that the FVL mutation, presumably due to thrombosis of placental vessels, may play a role in some cases of unexplained recurrent pregnancy loss and in certain obstetric complications (see Factor V Leiden and Thrombotic Risks Associated With Pregnancy and Factor V Leiden and Recurrent Pregnancy Loss ). Heterozygous carriers of FVL have been shown to have an overall 3- to 7-fold increased risk of venous thrombosis, while homozygotes have a 50- to 100-fold increased risk. 18,19,38,39 Despite the increase in risk for venous thrombosis, there is no evidence that heterozygosity for FVL increases mortality. 40 Multiple studies have evaluated the effect of FVL on the risk of VTE ,35,38,41,42 This mutation is present in approximately 10% to 20% of patients with a first VTE event without a family history of VTE, as illustrated by the following observations: The Physicians Health Study found a 12% incidence of heterozygosity for the FVL mutation in patients with a first confirmed DVT or pulmonary embolism compared to 6% in controls. 19 The incidence reached 26% in 31 men older than 60 years who had no identifiable precipitating factors. 19 The Leiden Thrombophilia Study, consisting of 471 patients younger than 70 years with a first confirmed DVT and 474 healthy control subjects, found a 21% incidence of APC resistance, compared to 5% in controls. 18,38 The incidence of heterozygosity (18% vs 3%) and homozygosity for FVL (1.5% versus 0%) was higher in the patients with thrombosis. 38 The relative risk for DVT was increased 7-fold for heterozygotes and 80-fold for homozygotes. In a study of 306 family members from 50 Swedish families, 40% of homozygotes had an episode of venous thrombosis by age 33 years, compared to 20% of heterozygotes and 8% of normals. 17 Despite the increase in thrombotic tendency, the risk of thrombosis in FVL homozygotes is significantly less than the risk in those with homozygous or doubly heterozygous protein C or protein S deficiencies. The latter conditions are rare, but the various case reports have been invariably associated with the severe syndrome of socalled neonatal purpura fulminans. 43 There are conflicting data as to whether the FVL mutation is associated with an increased risk of recurrent VTE. 42,44 48 In 2 series, for example, patients with FVL who had a first venous thrombotic event were more than twice as likely to have a recurrent episode than were those without the mutation during follow-up periods ranging from 5.7 to 8 years. 42,44 In contrast, 4 other studies found no difference in the incidence of recurrence between those with and without FVL In 2 recent reports, however, the risk of recurrence was significantly increased in FVL patients who also were heterozygous for the prothrombin gene mutation (relative risk 2.6 or 9.1), particularly in those in whom the first episode of DVT was spontaneous (relative risk 5.1 or 4.0). 46,49 The risk of recurrent VTE has also been shown to be 3-fold higher in homozygous carriers of FVL. 47 Factor V Leiden carriers with other coexisting thrombophilic defects may also have a higher risk of recurrence. For example, the observation that the risk of recurrent thrombosis is increased in patients with deficiencies of antithrombin, protein C, or protein S suggests that the risk of recurrence may also be increased when a heterozygous FVL mutation is combined with one of these anticoagulant protein deficiencies. 50,51 Similarly, since patients with antiphospholipid antibodies have an increased risk of recurrent VTE, heterozygous FVL carriers with antiphospholipid antibodies likely also have a higher risk of recurrence. 48,52 High plasma factor VIII levels are also a risk factor for recurrent as well as first VTE. 53,54 Other studies have demonstrated that elevated plasma concentrations of several other procoagulant factors, including fibrinogen, 55 factor IX, 56 factor X, 57 and factor XI, 58 confer an increased risk of VTE. Heterozygous FVL carriers with high levels of 1 or more of these procoagulant factors may then have an increased risk of recurrence, although this possibility has not been formally studied. Similarly, the risk of recurrence among symptomatic FVL heterozygotes who also carry the factor V HR2 haplotype (His1299Arg, a weak thrombotic risk factor) is unknown. 59,60 The lifetime probability of developing thrombosis ( 10%) and the severity of the thromboses are considerably less in heterozygotes with the FVL mutation than in patients with the less common inherited thrombophilias (ie, deficiencies of antithrombin, protein C, or protein S). This was illustrated in a study that compared the risk for thrombosis in individuals with inherited thrombophilia due to FVL or to antithrombin, protein C, or protein S deficiency in 150 pedigrees. 61 The lifetime probability of developing thrombosis compared to those with no defect was 8.5 times higher for carriers of protein S deficiency, 8.1 for antithrombin deficiency, 7.3 for protein C deficiency, and 2.2 for FVL. The vast majority of FVL heterozygotes ( 90%) will therefore not develop a symptomatic VTE event in their lifetimes. Based on these risks, consensus recommendations on the patient populations that should (and should not) be tested for FVL are defined in Table 3. COMBINED THROMBOPHILIC DEFECTS There appears to be an increased incidence of a second defect among thrombotic patients with FVL. This relationship has been described with deficiencies of protein C, 62,63 protein S, 64,65 antithrombin, 66 the prothrombin gene mutation, 67,68 and possibly homozygosity for the C677T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene (which is associated with an increase in the plasma homocyst[e]ine concentration). 68 A pooled analysis of 8 case-control studies comprising 2310 VTE cases and 3204 control subjects has recently found, for example, that patients carrying both the FVL and prothrombin G20210A mutations have an odds ratio for venous thrombosis of 20 (95% confidence interval [CI] 11 36) as compared to an odds ratio of 4.9 (95% CI ) for FVL alone and 3.8 (95% CI ) for prothrombin G20210A alone. 69 In addition, a study of 18 unrelated thrombosisprone families with inherited protein S deficiency found the factor V gene mutation in 39%. 65 In another report, which compared 113 patients with protein C deficiency and 104 healthy volunteers, the FVL mutation was much more common in those with protein C deficiency than in controls (15% vs 1%). 63 Carriers of 2 defects seem to be at a higher risk for thrombosis than their relatives with a single defect. In a review of 4 studies, approximately 75% of the family members who were carriers of 2 defects had experienced 1308 Arch Pathol Lab Med Vol 126, November 2002 Factor V Leiden Press et al

6 thrombosis compared with 10% to 30% of the carriers of a single defect. 70 The presence of 2 defects in these studies increased the thrombotic risk 3-fold above the risk of a single defect. The risk of thrombosis is also increased in patients with FVL and hyperhomocyst(e)inemia. In a large prospective cohort study, for example, the relative risk for idiopathic venous thromboembolic disease compared to patients with neither abnormality was 3.4 with hyperhomocyst(e)inemia, 2.3 with the FVL mutation, and 21.6 with both disorders. 71 INTERACTION OF FVL WITH ACQUIRED THROMBOPHILIC RISK FACTORS There is an important interaction of FVL with other risk factors for venous thrombosis, such as oral contraceptives and pregnancy (see Factor V Leiden and Oral Contraceptives ). Interestingly, the FVL mutation does not appear to add much additional risk to the underlying thrombotic risk in patients who have cancer, are undergoing surgery, or have pulmonary emboli. It is still unclear to what extent the FVL mutation adds to the overall thrombotic risk in patients undergoing orthopedic surgery. In a retrospective study of 825 patients undergoing hip or knee replacement surgery, the FVL mutation was not associated with a significantly increased risk of a venographically documented DVT during the immediate postoperative period. 72 The absolute incidence of DVT (often asymptomatic) was 31% in patients with the mutation and 26% in those without the mutation. In contrast, another recent study found that patients with APC resistance had a 5- fold increased risk of symptomatic postoperative VTE during the 2 months after elective hip replacement. 73 In addition, heterozygous FVL carriers who develop VTE have a significantly increased risk of having had surgery within 3 months preceding the thrombotic episode. One possible explanation for these discrepant results is that most asymptomatic thromboses after total hip or knee replacement resolve spontaneously without clinical sequelae. However, in patients with FVL, these initially small thromboses may propagate to occlude the vessel lumen and become symptomatic. The risk of developing isolated pulmonary emboli (ie, without concomitant DVT) in patients with FVL has been reported to be about one half (odds ratio 2.5) that of the risk of developing DVT with (odds ratio 5.2) or without (odds ratio 6.0) pulmonary emboli. 74,75 The cause of this interesting paradox is not known, but may be due to the lower incidence, in patients with FVL, of deep vein thrombi affecting the large, proximal iliofemoral vessels most often associated with the generation of pulmonary emboli. 76 FACTOR V LEIDEN AND CEREBRAL VEIN THROMBOSIS The FVL mutation occurs with increased frequency in patients with cerebral vein thrombosis (10% 20% in patients vs 2% 3% in control subjects) As with DVT, cerebral vein thrombosis occurs more frequently in young women who are taking oral contraceptives or who are pregnant or in the postpartum state. The use of oral contraceptives alone is a strong risk factor for cerebral vein thrombosis, and the addition of oral contraceptives to the presence of FVL results in a risk that exceeds the sum of the 2 separate risk factors. In a case-control study, for example, the estimated odds ratios for cerebral vein thrombosis were 10 for the use of oral contraceptives, 3 to 4 for hereditary prothrombotic disorders, and 34 for the presence of both risk factors. 80 FACTOR V LEIDEN AND ARTERIAL THROMBOSIS An association between FVL and arterial disease has not been well established. Although it is biologically plausible to postulate that FVL carriers with atherosclerotic vascular disease are at increased risk for arterial thrombosis, the weight of the current evidence does not support such an association Multiple studies did not find an increased prevalence of the mutation in patients with myocardial infarction or stroke. 19,85 91 Thus, routine anticoagulation is not recommended for FVL carriers with atherosclerotic arterial occlusive disease. However, among carriers with myocardial infarction or stroke, anticoagulation therapy for secondary prevention may be appropriate. 92 There may, however, be a small arterial thrombotic risk in both male and female FVL carriers that is amplified considerably when there are additional coronary risk factors. In a casecontrol study in young women (aged years), for example, the FVL mutation was associated with a 2.4-fold increase in risk of myocardial infarction after adjustment for age; this increase in risk was limited to current smokers. 93 A similar role for other major cardiovascular risk factors has been observed in men with a first myocardial infarction. 89 In another study, FVL was found in 12% of young patients (mean age 44 years) with myocardial infarction and normal coronary angiography, in 4.5% of ageand sex-matched patients with myocardial infarction and significant coronary artery disease (odds ratio 2.6, P.01), and in 5% of normal controls (odds ratio 2.9, P.01). 94 This finding supports the hypothesis that thrombosis plays a key role in this highly selected population. It has been suggested that FVL may be a more important contributor to cerebral infarction in children than in adults. 95 In a series of 26 such children, FVL was present in 6 children, 2 of whom also had protein C deficiency. 96 FACTOR V LEIDEN AND THROMBOSIS IN RENAL TRANSPLANT RECIPIENTS Renal transplant recipients carrying a mutant FVL allele have an approximately 4-fold increased risk of venous thrombotic events, 97,98 similar to the thrombotic risk in patients without kidney disease. As shown in several casecontrol studies of renal transplant recipients, those with FVL carry a significantly increased risk of early graft perfusion defects, early graft loss (within 7 days), acute allograft rejection, and primary allograft thrombosis In one such study, 100 the vascular rejection episodes in the FVL carriers were associated with allograft endothelialitis or fibrinoid vascular necrosis. Carriers of FVL also had a lower 1-year allograft survival rate (56%) than did noncarriers (76%). 100 Consistent with a role for hypercoagulability in long-term allograft survival and function, another case-control study of renal transplant recipients showed that those with thrombophilia (over half of whom were FVL carriers) had a shorter median graft survival (30 months) than those without laboratory-defined thrombophilia. 101 Given the higher risk for adverse renal allograft vascular events in FVL mutation carriers, a recent intervention study showed a 2.6-fold reduction in allograft thrombotic events in hypercoagulable patients (including those with FVL) receiving postoperative prophylactic anticoagulation. 102 Screening renal transplant recipients for Arch Pathol Lab Med Vol 126, November 2002 Factor V Leiden Press et al 1309

7 FVL (and other hypercoagulable states) particularly those with personal or familial thrombotic histories and/ or prior allograft failures may thus be an effective method to target high-risk patients for whom postoperative anticoagulation could provide a clinical benefit. ACTIVATED PROTEIN C RESISTANCE WITHOUT FVL Some small percentage of patients with APC resistance, identified using the first generation aptt-based assay, do not have an FVL mutation, as in the following examples. 1. Individuals with cerebrovascular disease have been described with APC resistance that is not due to the FVL mutation. 103,104 In one study, the investigators divided patients into 5 categories of responsiveness to APC as opposed to the usual practice of using a cutoff value for optimal separation of carriers and noncarriers of the mutation. 104 Statistical analysis showed that a low response to APC was associated with an increased risk of cerebrovascular disease, which was independent of the FVL mutation. 2. In a case-control study including 474 patients with first DVT and 474 age- and sex-matched control subjects, in which all carriers of the FVL mutation were excluded, a dose-response relationship was observed between the sensitivity for APC and the risk of thrombosis. 105 After correcting for confounding variables, a reduced response to APC remained a risk factor (odds ratio for the lowest quartile was 2.5). 3. In a study of more than participants who did not have FVL in the Vicenza Thrombophilia and Atherosclerosis Project, the adjusted odds ratio for development of VTE was 1.8 in those with phenotypic resistance to APC. 106 The clinical importance of documenting this type of non-fvl APC resistance is uncertain. The use of assays to identify these individuals is best restricted to thrombosis research centers. FACTOR V LEIDEN AND ORAL CONTRACEPTIVES The use of oral contraceptives substantially increases the risk of VTE in women with FVL. Factor V Leiden is found in 20% to 30% of women with a history of venous thrombosis during oral contraceptive use In the Leiden Thrombophilia Study, oral contraceptive use was associated with a 4-fold increase in risk of VTE. A heterozygous FVL mutation was associated with a 7-fold increase in risk. However, the risk of thrombosis was increased 35-fold in women with both risk factors. The corresponding thrombotic risk is increased more than 100- fold in homozygous carriers of FVL who use oral contraceptives. 110 These observations indicate that the combination of these 2 risk factors has a multiplicative, rather than an additive effect on the overall thrombotic risk. The evidence also suggests that women with inherited thrombophilic disorders such as FVL tend to develop thrombotic complications sooner, with a much higher risk of thrombosis during the first year of oral contraceptive use. 111 Data from the Leiden Thrombophilia Study also indicate that oral contraceptives containing the third generation progestagen desogestrel are associated with a 2-fold higher risk of VTE than second generation preparations, and the risk is especially high in carriers of FVL. The risk of thrombosis was increased 50-fold in FVL carriers who used third generation preparations containing desogestrel, compared to noncarriers not using oral contraceptives. 112 The synergistic interaction between FVL and oral contraceptives likely reflects the fact that both risk factors result in resistance to APC. Two studies have used a thrombin generation assay to demonstrate that plasma from women using oral contraceptives was substantially less sensitive to the anticoagulant effect of APC than plasma from nonusers. 113,114 Plasma from heterozygous FVL carriers using oral contraceptives showed even more profoundly reduced sensitivity to APC, in the range of that of women homozygous for FVL. The markedly increased risk associated with the combination of these 2 risk factors raises questions about the value of screening for FVL before prescribing oral contraceptives. Widespread screening of all women contemplating oral contraceptives is difficult to justify on a population level, given the unacceptably high cost-benefit ratio. Despite the marked increase in relative risk in FVL carriers using oral contraceptives, the absolute incidence of VTE is still low owing to the rarity of thrombosis in healthy young women. The combination of FVL and oral contraceptives results in an additional 28 venous thrombotic events per women per year. 110 Assuming that 2% of thromboembolic episodes are fatal, it is estimated that women would have to be screened in order to identify carriers of FVL, who would all have to be denied oral contraceptives to prevent 1 thromboembolic death. 115 Another study, assuming a lower 1% mortality rate from VTE in young women, estimated that nearly 2 million women would have to be screened to identify FVL carriers to prevent 1 death from VTE. 116 Estimates of the cost to prevent 1 death from VTE by universal screening range from $74 million to more than $300 million. 116,117 Other arguments against universal screening are that it would deny the most effective form of contraception to 5% of all women, which could result in a large number of unplanned pregnancies, also associated with an increased thrombotic risk. The cost of such a widespread screening program would be prohibitive and could result in a large number of asymptomatic women being labeled with a genetic disorder. Although decisions regarding screening and the use of oral contraceptives or hormone replacement therapy (HRT) should be made on an individual basis, taking into account the personal and family history and coexisting risk factors, routine FVL screening of all oral contraceptive users cannot be justified. FACTOR V LEIDEN AND HRT AND SELECTIVE ESTROGEN RECEPTOR MODULATORS At least 40% of postmenopausal women in the United States are currently using HRT. The dose of estrogen in HRT is one sixth that found in modern low-dose oral contraceptives. 118 Physiologic replacement doses of estrogen were previously thought to be associated with little or no increase in thrombotic risk. However, at least 7 recent studies consistently found a significant 2- to 4-fold increase in relative risk of VTE in current HRT users compared to nonusers. 4,5, The limited data available suggest that selective estrogen receptor modulators, such as tamoxifen and raloxifene, are also associated with a similar increase in thrombotic risk Most of the observational studies of HRT excluded women with known thrombophilia or other thrombotic risk factors. In 2 recent studies, however, the combination 1310 Arch Pathol Lab Med Vol 126, November 2002 Factor V Leiden Press et al

8 of HRT use and FVL (or APC resistance) was associated with a 13-fold 127 or 15-fold 128 increase in relative thrombotic risk, compared to women without either risk factor. To date, there have been no other studies of the potential increase in thrombotic risk in women with FVL who use HRT. However, given the interaction between estrogens and FVL, it is likely that carriers of the mutation are at a higher risk for thrombotic complications associated with HRT. Several cases of tamoxifen-associated thrombosis in women with FVL have been reported. 129 In light of the increasing use of selective estrogen receptor modulators in the treatment and prevention of breast cancer and osteoporosis, it is also likely FVL will be shown to increase the risk of selective estrogen receptor modulator associated thrombosis in future studies. FACTOR V LEIDEN AND THROMBOTIC RISKS ASSOCIATED WITH PREGNANCY The risk of VTE is 5- to 6-fold higher during pregnancy than in nonpregnant women of similar age, and it is even higher during the postpartum period. There is convincing evidence linking FVL to an increased risk of VTE during pregnancy and the postpartum period. Resistance to APC using a first generation assay was found in up to 60% of women with a history of VTE during pregnancy, compared to 10% of nonpregnant control women. 109 The FVL mutation was found in 20% to 46% of women with pregnancy-associated VTE in retrospective case series and case-control studies. 108, The available data suggest that FVL is associated with a 7- to 16-fold increased thrombotic risk during pregnancy and the puerperium. In one recent study, FVL was found in 44% of women with a history of VTE during pregnancy compared to 8% of matched control subjects, and it was associated with a 9- fold increase in thrombotic risk. 130 The relative risk of thrombosis during pregnancy was increased more than 100-fold in women with both FVL and the prothrombin G20210A mutation, illustrating the dramatic increase in overall risk when thrombophilic mutations are combined. Women with homozygous FVL also have a higher risk of pregnancy-related VTE. In one study of family members of symptomatic probands with FVL, venous thrombosis occurred in 16% of pregnancies in homozygous women compared to 0.5% of those in unaffected relatives, conferring a 40-fold increase in relative thrombotic risk. 134 Although FVL increases the risk of VTE during pregnancy and the puerperium, the true risk in asymptomatic carriers is unknown. Estimates of thrombotic risk are based primarily on retrospective case-control and cohort studies that may overestimate the risk in asymptomatic carriers. Several recent studies provide an estimate of the absolute risk of pregnancy associated VTE in FVL carriers. One prospective study screened unselected pregnant women for FVL and followed them throughout pregnancy. Thrombotic complications occurred in only 1.1% of FVL carriers. 135 In a large retrospective study of more than unselected pregnant women, the estimated risk of VTE during pregnancy and the puerperium in FVL carriers was in the range of 1 in 400 to 500 pregnancies. 136 Another retrospective study calculated a similar probability of thrombosis of 1 in 400 FVL pregnancies. 130 The results of these studies suggest that the absolute incidence of FVL-related thrombosis during pregnancy is low, and they do not support routine screening of all pregnant women for this mutation. It has been estimated that if screening all pregnant women resulted in routine prophylactic anticoagulation of FVL carriers, the number of cases of fatal bleeding would equal and possibly exceed the number of fatal pulmonary emboli prevented. 115 FACTOR V LEIDEN AND RECURRENT PREGNANCY LOSS Serious obstetric complications occur in 1% to 5% of pregnant women and include recurrent pregnancy loss (RPL), preeclampsia, fetal growth retardation, and placental abruption. Recurrent pregnancy loss is a well-established complication of the antiphospholipid antibody syndrome and is thought to result from thrombosis of placental vessels, often with evidence of placental infarction. More recently, inherited thrombophilic defects including FVL have been linked to RPL and other obstetric complications. At least 16 case-control studies found a high prevalence of FVL in women with unexplained RPL (up to 30%) compared to 1% to 10% of control subjects (odds ratios ranging from 2 to 5. 35, The results were consistent despite differences in study populations and selection criteria. Six other case-control studies found no association between FVL and RPL. 37, These latter studies were smaller and most included women with common first trimester fetal losses (often due to non thrombophilia-related factors). Three retrospective cohort studies found that FVL carriers have a significantly increased risk of RPL. 37,157,158 In one study of a large cohort of women with thrombophilia (including 141 with FVL), FVL carriers had a 2-fold increased risk of stillbirth, but there was no increased risk of miscarriage before 28 weeks. 158 Another study found that FVL carriers had a 2-fold increased risk of fetal loss by 20 weeks. Women with homozygous FVL had a 2-fold higher risk of fetal loss than heterozygous carriers. Recurrent loss was also more common in FVL carriers (odds ratio 2.6). 157 Factor V Leiden carriers who are family members of probands with the mutation have an approximately 2-fold increased risk of fetal loss after the first trimester. 37 In one small prospective study, miscarriage after the first trimester occurred in 11% of FVL carriers compared to 4.2% of women with a normal genotype. 159 Although most pregnancy losses occur in the first trimester, women with thrombophilia have the highest risk of loss in the second and third trimesters. Four studies found that FVL carriers have a significantly higher risk of late pregnancy loss than early first trimester loss. 37,140,143,158 In some studies, FVL was significantly associated with only second or third trimester losses. 37,140,158 One possible explanation is that late pregnancy losses may reflect thrombosis of placental vessels, in contrast to first trimester losses, which are more commonly due to other causes. In several studies, the majority of placentas from women with FVL and late fetal loss had evidence of thrombotic vasculopathy or infarction. 141,142 In another study, FVL was found in 42% of a large cohort of placentas with major infarction. 144 The frequent finding of placental infarction suggests that RPL associated with FVL is due to thrombosis. Although preeclampsia, fetal growth retardation, and placental abruption are also thought to involve impaired placental perfusion, their association with FVL remains controversial, with conflicting results from different studies. In one recent study, FVL was found in 20% of the women with preeclampsia, placental abruption, fetal Arch Pathol Lab Med Vol 126, November 2002 Factor V Leiden Press et al 1311