Many assays used in the laboratory evaluation of hypercoagulability are affected by concurrent clinical conditions and medications. Whenever possible, a thorough clinical history including site of thrombosis, previous bleeding or thrombotic events, other medical disorders (i.e., liver, autoimmune, or cardiovascular disease), pregnancy, medications (including but not limited to anticoagulants), and family history should be elicited. These are essential for appropriate test selection and interpretation of results; sources of interference for specific assays are described in the following sections. Tests should be performed at least 4-6 weeks after an acute thrombotic event or discontinuation of anticoagulant/thrombolytic therapies including warfarin, heparin, direct thrombin inhibitors (DTIs), direct factor Xa inhibitors, and fibrinolytic agents [1, 4, 5]. If abnormal results are found during acute illness or anticoagulant therapy, testing should be repeated in a new specimen when the patient is stable and after anticoagulant therapy is discontinued. Alternatively, thrombophilia testing may be delayed until acute clinical conditions have subsided. The exception is DNA analysis for genetic mutations, which is not generally affected by other medical issues or anticoagulant therapy.

No single laboratory test is yet available that can identify all hypercoagulable defects. Selection of the most informative tests may differ depending on location and type (venous or arterial) of thrombosis. Fig. 1 outlines a testing algorithm to maximize diagnostic potential in patients with thrombophilia while avoiding unnecessary and potentially expensive tests. Testing should be performed in a step-wise manner beginning with high-yield screening tests followed by appropriate specific confirmatory tests. These comprehensive panels generate multiple test results that can each be affected by a variety of clinical conditions and drugs. Comprehensive narrative interpretation by coagulation specialists is necessary to synthesize test results, correctly interpret them according to the patient's clinical condition, and provide appropriate guidance to clinicians [2, 7]. In some patients with thrombophilia, it may be best to test for all recognized hereditary risk factors, both common and uncommon [5, 7, 8].

Antiphospholipid syndrome (APS) is the most common cause of acquired thrombophilia. Antiphospholipid antibodies (APAs) are acquired autoantibodies directed against phospholipid-protein complexes and are present in 3-5% of the general population. APAs are associated with increased risk of both arterial and venous thrombosis and recurrent pregnancy loss [9, 10]. APAs can arise spontaneously (primary) or in association with another condition (secondary). Also known as lupus anticoagulants (LA) because of their prevalence in patients with systemic lupus erythematosus (SLE), APAs are extremely heterogeneous and can be directed against a wide variety of anionic phospholipids, including cardiolipin, beta 2 glycoprotein 1 (B2GP1), and cell-membrane phosphatidylserine [3, 11].

Diagnosis of APS requires clinicopathologic correlation because both clinical (either proven vascular thrombosis or pregnancy morbidity) and laboratory criteria must be met. Laboratory diagnostic criteria include positive testing for 1 of the following on 2 or more occasions, at least 12 weeks apart: (1) lupus anticoagulant; (2) anticardiolipin antibodies (IgG or IgM) in medium or high titer; or (3) B2GP1 antibodies (IgG or IgM) in medium or high titer [5, 10, 11, 12]. A repeated positive test after a ≥12-week interval is required for diagnosis because transient low-level increases in APA occur in a variety of clinical conditions, including acute phase response, and may not confer increased risk of thrombosis.

Protein C is a vitamin K-dependent serine protease primarily synthesized in an inactive form by the liver [3]. Activation of protein C (APC) requires interaction with the thrombin-thrombomodulin-endothelial protein C receptor complex. Activated protein C regulates thrombin generation by degradation of activated coagulation factors Va and VIIIa in the presence of its cofactor protein S [1]. APC resistance (APC-R) is observed in approximately 20% of patients with a first episode of DVT and 50% of familial thrombosis. More than 90% of APC-R patients have a point mutation in the factor V gene, known as FVL mutation [2, 23, 24]. The FVL mutation (G1691A) results in substitution of glutamine for arginine at position 506 (R506Q); this arginine is 1 of the 3 arginine sites (R306, R506, and R679) cleaved by APC [1]. FVL is present in a heterozygous form in approximately 3-5% of the general Caucasian population and is rare in African, Australian, and South Asian populations [2, 25]. The FVL mutation is the most commonly known hereditary risk factor for venous thrombosis (VT); however, risk for arterial thrombosis remains unclear. VT risk is increased 4- to 8-fold in FVL heterozygotes and 80-fold in homozygotes [6, 26]. Risk of pulmonary embolism (PE) may not be as high as risk of DVT [23]. Thrombotic risk is further increased in the presence of a second risk factor: female FVL heterozygotes using oral contraceptives (OCPs) appear to have a 30- to 60-fold increased risk of thrombosis. Other FV mutations occur but are much less common, including FVR2 haplotype (H1299R), FV Liverpool (I359T), FV Cambridge (R306T), and FV Hong Kong (R306G). These affect APC-R and thrombotic risk [27, 28, 29].

Acquired APC-R can be caused by the development of autoantibodies against factor V following exposure to bovine thrombin or with untreated hematological malignancies, LAs, pregnancy, OCPs, active thrombosis, elevated factor VIII, and mutations in the factor VIII gene [2, 4].

Laboratory assays for APC-R and FVL mutation include functional assays and genotyping. The functional APC-R assay is based on prolongation of aPTT by degradation of factors Va and VIIIa by exogenously added APC. The ratio of aPTT in patient plasma and normal plasma before and after adding APC is calculated. This ratio in normal individuals is ≥2.0; in FVL heterozygotes, 1.5-2.0; and in homozygotes, <1.5. Each laboratory should determine its own cutoff for an abnormal result [4, 18]. Elevated factor VIII, low protein S (<20%), and causes of prolonged baseline aPTT (heparin, warfarin, DTI, LA, liver dysfunction/low factor levels) can cause a falsely low APC-R ratio. A second-generation assay includes dilution of patient plasma with factor V-deficient plasma (also containing heparin neutralizer) and provides higher sensitivity and specificity. This assay is less affected by active thrombosis, surgery, inflammatory condition, heparin, or warfarin [30].

Identification of FVL mutation as the cause of APC-R is confirmed by DNA analysis such as PCR-restriction fragment length polymorphism or allele-specific PCR genotyping. A non-PCR-dependent, simple microtiter plate-based Invader assay using fluorescence resonance energy transfer detection shows a reliable detection rate for FVL mutation. However, use of specific primers limits this test to detecting only specific mutations (i.e., FVL), and will not detect other FV mutations. In general, a cost-efficient functional APC-R assay is recommended as an initial screen, with DNA analysis for FVL mutation added for confirmation in individuals with abnormal results [31, 32].

The prothrombin gene G20210A mutation is a gain of function mutation. The mutation occurs in an intron near the 3' end of the gene and alters 3' end processing and/or enhances translation efficiency, resulting in increased prothrombin (factor II) levels [33]. However, the exact mechanism of how increased prothrombin gene expression causes hypercoagulability remains unclear. The prothrombin gene G20210A mutation is the second most common hereditary risk factor for VT [1, 2]. Prevalence varies by ethnicity; 2-4% of Europeans carry the mutation, but it is rare in Asians, Native Americans, and Africans [16, 34, 35]. This mutation is present in approximately 1-3% of the general population, 5-10% of patients with VT, and up to 20% of patients with VT from thrombophilic families. Heterozygous individuals show a 3-fold increased VT risk. However, VT risk will be drastically increased when the patients carry additional inherited or acquired risk factors [2, 4, 36].

Genetic testing to detect the G20210A mutation can be performed by PCR-based methods. Newer assays based on various PCR methods coupled with fluorescence polarization methods or the Invader assay can be performed on automated platforms. DNA microarray technology can detect multiple genetic markers simultaneously with a relatively low cost as a single test compared with conventional DNA assays [32, 33]. However, these tests require expensive equipment and skilled personnel and may require reflex confirmatory assays (sequencing) if there is an ambiguous or atypical result [32].

Protein C deficiency occurs in 0.14-0.50% of the general population and 1-3% of patients with VTE [4, 18]. It is inherited in an autosomal dominant fashion; heterozygotes show functional protein C levels of 40-65% of normal and have a 7-fold increase in risk for VT [35]. The first thrombotic event usually presents at 10-50 years of age [4]. Protein C deficiency also carries increased risk for warfarin-induced skin necrosis. Homozygotes are very rare and can present with neonatal purpura fulminans or disseminated intravascular coagulation (DIC).

Protein C assays measure activity (functional) or antigen quantity (immunological). A functional protein C assay, either clot-based or chromogenic, is usually performed first; if the result is low, an antigenic protein C assay is performed to determine whether the protein defect is quantitative (type I) or qualitative (type II). Type I protein C deficiency is characterized by reduced functional activity and antigen levels and is much more common (75-80%) than type II deficiency (20-25%), which is further subdivided into types IIa (24.5%) and IIb (0.5-1.0%). Both IIa and IIb result in reduced activity with normal antigen levels. If only a quantitative antigenic assay is used, type II deficiency cannot be detected [1, 2]. Clot-based functional protein C assays can detect both types I and II deficiencies but can give falsely increased results with anticoagulant therapy, LAs, and FVL mutation, and falsely decreased results with elevated factor VIII levels (particularly >250%) or low protein S. The chromogenic assay is less affected by interfering substances and is more reproducible; however, it only assesses alterations in the activation and active sites of the protein (type IIa defects) and cannot detect defects in other sites (protein S, surface, or substrate binding sites) and therefore can overlook the rarer type IIb deficiencies [4, 7, 18, 37]. Functional assays, either chromogenic or clot-based, are recommended as initial screens with antigenic assays performed if results are abnormally low.

Acquired deficiency is more common than hereditary forms and must be excluded before making a diagnosis of hereditary protein C deficiency. Because protein C is synthesized in hepatocytes and is vitamin K-dependent, both liver dysfunction and vitamin K deficiency (including warfarin therapy) decrease protein C levels. Protein C has a short half-life (6-8 hours), and levels decrease more rapidly than other coagulation proteins (including protein S and antithrombin) in liver disease and warfarin therapy/vitamin K deficiency. Conversely, protein C levels rapidly normalize after discontinuation of warfarin or correction of vitamin K deficiency; however, levels should not be measured for at least 10 days after warfarin discontinuation. Protein C levels are lowered in recent or current thrombosis, DIC, L-asparaginase therapy, and nephrotic syndrome and during the intra- or immediately postoperative period; neonates also have relatively low protein C levels (17-53%). OCP use and pregnancy can increase protein C levels [2, 7, 38]. Abnormal protein C assays should be repeated after any such conditions have resolved to confirm results.

Protein S is a vitamin K-dependent glycoprotein that acts as a cofactor to protein C, accelerating proteolysis of factors Va and VIIIa by approximately 2-fold [39]. Approximately 60% of protein S in the plasma is bound noncovalently to C4bBP in plasma with high affinity; the remaining free (unbound) protein S is the predominant active form. Recent studies have shown that protein S also exerts its own anticoagulant activity by direct binding of factors V, VIII, and X, and appears to act as a cofactor for the tissue factor pathway inhibitor, which results in inhibiting tissue factor-mediated factor X activation [39, 40]. Hereditary protein S deficiency is transmitted in an autosomal dominant fashion and occurs in 0.2-0.5% in the general population and in 1-3% of patients with first VT [7, 37, 41]. Functional protein S levels range 20-64% in heterozygous patients [42]. Homozygous patients typically present as newborns with purpura fulminans and DIC.

There are 3 types of protein S deficiency. Types I and III are quantitative deficiencies with both low free protein S antigen and low protein S activity, and account for 95% of cases. Type I deficiency shows low total (free+C4bBP-bound) protein S antigen level, whereas type III shows normal total protein S levels. Type III deficiency may be related to excess binding of protein S to C4bBP. Type II deficiency is a qualitative defect with low protein S activity and normal antigenic (free and total) protein S levels [35, 43, 44].

Functional (clot-based) protein S assays measure activity, and immunologic methods (monoclonal antibody-based enzyme immunoassay and immunoturbidimetric assay) measure antigenic levels of free or total protein S. Clot-based functional assays are sensitive for all types of deficiency and are used by some laboratories as an initial screening test, similar to the algorithm for protein C. However, functional protein S assays are not specific; potential sources of interference include APC-R (usually from FVL mutation) and LAs. Despite the fact that using an antigenic test as an initial assay may miss type II protein S deficiency, some guidelines recommend antigenic tests as a screen with functional tests performed only on abnormal results [45].

As with protein C deficiency, acquired causes of protein S deficiency are more common and should be excluded before making a diagnosis of hereditary deficiency. Protein S is decreased in conditions that decrease protein C (see the previous section). Protein S is also decreased during the acute phase response because C4bBP is an acute phase reactant; increased C4bBP lowers both protein S activity and free antigen. Protein S is decreased with elevated factor VIII (>250%), and infectious and autoimmune conditions such as HIV infection and inflammatory bowel disease [4, 18, 41]. Levels are usually lower in women, especially during hormone replacement therapy, OCP use, and the second or third trimester of pregnancy [1]. Abnormal results should be confirmed after any such conditions are resolved.

Antithrombin (half-life: 2-3 days) is a glycoprotein of the serine protease inhibitor (serpin) family that primarily inactivates activated thrombin (factor IIa) and factor Xa, and to a lesser extent, factors IXa, XIa, and XIIa. Antithrombin acts as a "suicide inhibitor" by forming 1:1 covalent complexes between antithrombin and serine proteases; inhibitor activity is greatly accelerated by interaction with heparin. Although synthesized in the liver parenchyma, antithrombin is not vitamin K-dependent [46, 47].

Antithrombin deficiency is inherited in an autosomal dominant fashion and associated almost exclusively with VT. Prevalence rates are approximately 0.05-0.1% in the general population. Estimated annual incidence of a first episode of VTE in heterozygotes is 1.0-2.9% annually in retrospective studies [18, 47]. Risk of thrombosis appears to be higher in antithrombin deficiency than protein C or protein S deficiency, APC-R, or prothrombin gene G20210A mutation and thus has the highest VTE risk among known hereditary thrombophilias. The homozygous state is almost universally fatal in utero. Functional antithrombin levels in heterozygous individual ranges 35-70% [4, 8, 48, 49]. Besides presenting as DVT or PE, VTE from antithrombin deficiency can occur in unusual sites, such as cerebral sinuses and the mesenteric, portal, and renal veins. The first event occurs at a young age (<50 years) but is uncommon during the first 2 decades of life, and may or may not follow a provocative event. Approximately 58% of first events occur spontaneously, whereas 42% are associated with a transient, potentially preventable, risk factor. Patients with concurrent defects such as FVL mutation are associated with higher risk of VTE at younger ages (median: 16 years) [41, 50].

Antithrombin assays measure both functional activity (usually by chromogenic methods) and antigen quantity (enzyme immunoassays and immunoturbidimetric methods). If the functional level is normal or elevated, antithrombin deficiency is unlikely. Low results should be confirmed, and both functional and antigenic levels should be tested on the new specimen to determine the type of deficiency [4, 18].

Type I antithrombin deficiency is a quantitative defect showing proportionately reduced (approximately 50%) functional and antigenic levels. Type II deficiency is a qualitative defect, resulting in lower activity than antigen. Type II deficiencies are further classified by antithrombin mutation site. However, subclassification is generally not clinically necessary because anticoagulant therapy does not differ between types [41, 46, 47, 49].

Acquired antithrombin deficiency must be excluded before making a diagnosis of hereditary deficiency and can be caused by drugs such as heparin or _L-asparaginase. Other causes of low antithrombin include reduced synthesis (liver disease) or increased loss in nephrotic syndrome, protein losing enteropathy, DIC, sepsis, burn, trauma, hepatic venoocclusive disease, thrombotic microangiopathies, cardiopulmonary bypass surgery, hematomas, or metastatic tumors. Activity can be reduced by up to 30% during full-dose unfractionated heparin therapy, but not low-molecular-weight heparin therapy, and levels normalize when heparin is discontinued. Antithrombin can also be low in premenopausal women, OCP use, and pregnancy. As with low protein C and protein S, confirmatory testing should be repeated on a new specimen after any potential confounding conditions have resolved [4, 7, 48, 51].

Homocysteine is an intermediate amino acid produced by demethylation of methionine via methylenetetrahydrofolate reductase (MTHFR) in the folate cycle. Homocysteine metabolism requires vitamin B6, vitamin B12, and folate. Hyperhomocysteinemia is associated with increased risk of VTE, coronary heart disease, acute myocardial infarction, peripheral artery disease, stroke, aneurysm, migraine, hypertension, male infertility, risk for offspring with neural tube defect, and recurrent pregnancy loss. Acquired hyperhomocysteinemia can be caused by vitamin B6, vitamin B12, or folate deficiency; renal failure; hypothyroidism; rheumatoid arthritis; and certain drugs such as methotrexate, niacin, anticonvulsant, theophylline, L-dopa, thiazide, cyclosporine A, or phenytoin [4, 52, 53].

Hereditary hyperhomocysteinemia is caused by polymorphisms in an enzyme necessary in homocysteine conversion pathways. Homozygous alterations of the MTHFR gene are present in 10-13% of the population, whereas heterozygous alterations are found in 30-40% [52]. Two commonly recognized polymorphic variants in the MTHFR gene are (1) the "thermolabile" c.665C->T (p.Ala222Val), historically referred to as c.677C->T, and (2) c.1286A->C (p.Glu429Ala), also known as c.1298A->C. Meta-analyses of 2 common polymorphisms have found a weak association with risk of thrombosis [7, 54, 55, 56]. Homozygosity for MTHFR c.665C->T is associated with an approximately 25% increase in plasma homocysteine. Patients with elevated homocysteine and the MTHFR c.665C->T and/or c.1286A->C mutations, however, may be at mildly increased risk for both VTE (odds ratio: 1.27) and recurrent pregnancy loss (pooled risk: 2.7) [56]. Lowering homocysteine levels using vitamin B6, vitamin B12, or folate has not been proven to reduce thrombotic risk. Given the modest increase in thrombophilic risk and lack of evidence of therapeutic benefit, screening homocysteine levels in healthy individuals and testing for MTHFR polymorphisms are not currently suggested [4, 38, 56].

Several studies have demonstrated an association between elevated factor VIII and increased risk of thrombophilia, partly due to factor VIII-mediated enhancement of thrombin generation [57, 58, 59, 60]. Although no genetic variations in the factor VIII gene have been identified, levels appear to be higher in African-Americans and lower in individuals with blood group O. Factor VIII can be elevated during acute phase reactions, elevated estrogen, pregnancy, or after aerobic exercise. The prevalence of elevated factor VIII among patients with VT is 20-25% [57, 60, 61]. Whether factor VIII elevation directly contributes to increased thrombophilic risk remains unclear; however, studies show that persistent factor VIII level >150% (or >90th percentile) in the absence of acute phase reactions, elevated estrogen levels, or recent exercise is an independent risk factor for thrombophilia [61, 62, 63].

Factor VIII activity can be measured by aPTT-based clotting assay or chromogenic assay, and antigen quantitation can be accomplished using ELISA. Factor VIII measurement should be postponed until at least 6 months after an acute thrombotic event and 6 weeks after giving birth and should be repeated after 3-6 months to confirm persistent elevation [2, 63].

Dysfibrinogenemia describes a heterogeneous group of disorders resulting in structurally and functionally altered fibrinogen. It can cause bleeding, venous or arterial thrombosis, or both. The prevalence of dysfibrinogenemia in patients with VT is approximately 0.8% [64, 65, 66]. Although the exact mechanism of thrombosis is unknown, it may be related to increased fibrin formation and/or impaired fibrinolysis. Patients with dysfibrinogenemia may have prolonged prothrombin time (PT), thrombin time, and reptilase time; decreased functional fibrinogen; and normal to elevated immunologic fibrinogen. The ratio of functional fibrinogen activity to immunologic fibrinogen antigen will be decreased [2, 7, 66, 67].

The most commonly used functional assay is the Clauss method. Acquired fibrinogen deficiency can be caused by liver disease, consumptive states such as placental abruption or DIC, or fibrinolytic therapy. Because fibrinogen is an acute phase reactant, testing should be delayed at least 6 months after acute thrombosis [67].