In most circumstances, a functional assay of plasma APC resistance is the preferred initial test. In the original assay, aPTT was measured before and after addition of a standardized amount of APC that was sufficient to prolong the aPTT about 2-fold. The result was usually expressed as the APC-R ratio (defined as the ratio of the aPTT clotting times in the presence and absence of APC). An APC-R ratio of less than 2.0 was indicative of APC-R. About 95% of patients with APC-R were carriers for Factor V Leiden. Although specific, the original APC-R assay was relatively insensitive. Factor V Leiden carriers can have an APC-R ratio as high as 2.9, and the assay did not reliably distinguish heterozygotes from homozygotes. Moreover, the original assay was uninterpretable if the baseline aPTT was prolonged due to warfarin or heparin anticoagulation, factor deficiency, or a lupus anticoagulant or other specific factor inhibitor.
A modified (second-generation) APC-R functional assay that overcomes the limitations of the original assay is now widely available and FDA approved. In this assay, the patient's plasma is first mixed with F5-deficient plasma that contains a heparin neutralizer. The addition of the F5-deficient plasma corrects for deficiencies of other coagulation proteins and may dilute the effect of some lupuslike anticoagulants. The modified assay is essentially 100% sensitive and specific for Factor V Leiden and accurately distinguishes heterozygotes from homozygotes. In addition, the second-generation assay is unaffected by heparin or warfarin anticoagulation. However, the assay may still be uninterpretable if the baseline aPTT (after mixing with F5-deficient plasma) is still prolonged due to a lupus anticoagulant or specific factor inhibitor. Moreover, the assay will miss patients with acquired APC-R. Each laboratory must determine its own normal range for this test.
Direct DNA testing for Factor V Leiden is widely available for diagnosis. Commonly used molecular methods use PCR amplification of the region surrounding the mutation followed by restriction enzyme digestion (PCR-RFLP), allele-specific PCR amplification, or allele-specific hybridization. Semiautomated assay methods include fluorescence detection of PCR products with allele-specific hybridization probes, and non-PCR signal amplification methods based on either enzymatic hybridization mismatch recognition using fluorescent allele-specific probes or linked fluorescent allele-specific pyrophosphorolysis-kinase reactions.22 Each laboratory must insure that its method can distinguish Factor V Leiden from an uncommon polymorphism at nucleotide 1696 (A^G). In 2003, Roche Diagnostics Corporation (Indianapolis, IN)
obtained FDA approval for in vitro diagnostic test kits for Factor V Leiden and the prothrombin 20210G^A gene mutation.
Recommendations for Clinical Use of Factor V Leiden Testing
Testing for Factor V Leiden is recommended for patients with the following events:22
• a history of recurrent VTE;
• a first VTE without clinical risk factors at any age;
• a first VTE at an unusual anatomic site such as the cerebral, mesenteric, portal, or hepatic veins;
• a first VTE at any age in a subject with a first-degree family member with a VTE before 50 years of age;
• a first VTE related to pregnancy, the puerperium, oral contraception, or hormone replacement therapy;
• unexplained pregnancy loss during the second or third trimester.
Testing for Factor V Leiden is controversial for the following individuals:
• young women smokers (age <50 years) with a myocar-dial infarction;
• older patients (age >50 years) with a first provoked VTE in the absence of cancer or an intravascular device;
• patients with a first VTE related to SERM therapy;
• selected cases of women with unexplained severe preeclampsia, placental abruption, or intrauterine growth retardation.
After appropriate counseling, testing for Factor V Leiden also may be indicated in asymptomatic adult family members of probands with known Factor V Leiden mutations, especially those with a strong family history of VTE at a young age (<50 years), and asymptomatic female family members that are pregnant or are considering oral contraceptives or pregnancy.
Factor V Leiden testing is not recommended for the following purposes:
• general population screening;
• routine initial test during pregnancy;
• routine initial test prior to or during oral contraceptive use, hormone replacement therapy, or SERM therapy;
• prenatal test, newborn initial test, or as a routine test in asymptomatic prepubescent children;
• routine initial test in patients with arterial thrombotic events.
However, Factor V Leiden testing may be appropriate for patients with unexplained arterial thrombosis without atherosclerosis or for young patients who smoke.
Individuals who are heterozygous or homozygous for Factor V Leiden with a first lifetime deep vein thrombosis or pulmonary embolism should be treated in standard fashion, initially with heparin (either unfractionated or low-molecular-weight heparin), followed by oral anticoagulation with a vitamin K antagonist (target INR 2.5; therapeutic range 2.0-3.0).22 In general, 3 to 6 months of oral anticoagulation therapy is recommended after a first lifetime VTE for patients with Factor V Leiden, especially if the event was associated with a transient clinical risk factor (e.g., surgery, oral contraceptive use, pregnancy, or the puerperium). The need for lifelong anticoagulation after a first episode of VTE for patients with Factor V Leiden has not been established by appropriate clinical trials. Therefore, indefinite anticoagulation should be recommended only after careful consideration of the risks and benefits. Indefinite anticoagulation may be recommended for patients with Factor V Leiden who have an idiopathic or life-threatening VTE (especially in the presence of reduced cardiopulmonary functional reserve), have more than one hereditary thrombophilia, are homozygous for a hereditary thrombophilia, or have additional persistent clinical risk factors (e.g., malignant neoplasm, serious neurologic disease with extremity paresis, antiphospholipid antibodies). Hereditary thrombophilia patients (or any patient) with recurrent unprovoked VTE should be considered for indefinite anticoagulation therapy.
Women with Factor V Leiden and a history of unprovoked VTE should receive prophylactic anticoagulation with heparin or low-molecular-weight heparin during pregnancy and for at least 6 weeks postpartum. Routine anticoagulation therapy is not recommended for individuals with Factor V Leiden who also have atherosclerotic arterial occlusive disease. However, for patients with myocardial infarction or stroke and Factor V Leiden, anticoagulation therapy for secondary prevention may be appropriate.
Anticoagulation therapy is not recommended for asymptomatic individuals with Factor V Leiden. Factor V Leiden carriers (with or without previous VTE) should receive appropriate prophylaxis when exposed to risk factors. Standard prophylaxis recommendations are sufficient for most types of surgery. A possible exception is an asymptomatic Factor V Leiden carrier undergoing hip-replacement surgery, who might be at increased risk of symptomatic VTE for several weeks after surgery. These patients should receive extended out-of-hospital prophylaxis, especially in association with obesity or prolonged immobilization.
Prophylactic anticoagulation is not routinely recommended in pregnant Factor V Leiden carriers with no history of thrombosis. Decisions about anticoagulation should be individualized based on the genotype (heterozygous or homozygous) and coexisting risk factors. Asymptomatic women who do not receive anticoagulation should be followed closely throughout pregnancy and given prophylaxis during the puerperium.
PROTHROMBIN 20210G^A MUTATION Molecular Basis of Disease
The prothrombin (Pt) 20210G^A mutation is a relatively common polymorphism that affects the 3'-terminal nucleotide of the prothrombin gene 3'-untranslated region.25 Individuals with this polymorphism have increased plasma prothrombin levels. The Pt 20210G^A mutation causes a gain of function due to increased recognition of the polyadenylation cleavage signal, increased 3' end RNA processing, mRNA accumulation, and increased protein synthesis.26 A family-based study has shown close linkage between a quantitative trait locus determining plasma prothrombin activity and the Pt 20210G^A mutation.27 Plasma prothrombin concentration is a major determinant of plasma thrombin generation potential. Thrombin generation is increased in the plasma of heterozygous and homozygous Pt 20210G^A mutation carriers. High plasma prothrombin levels also inhibit APC-mediated in-activation of procoagulant F5a, further augmenting thrombin production.
Based on haplotype analyses, the Pt 20210G^A mutation also appears to be a founder mutation, arising 20,000 to 30,000 years ago, and after the divergence of Africans from non-Africans and of Caucasoid from Mongoloid subpopulations. In Europe, the overall carrier frequency is 2%, ranging from 1.7% in northern Europe to 3.0% in southern Europe. In the United States, the estimated overall carrier frequency is 1% to 2%. The mutation is uncommon among African Americans and rarely seen among Asian Americans and Native Americans.28
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