Thrombophilia is characterized by hypercoagulability and an increased propensity for thrombosis. Almost 2 million Americans succumb annually to a thromboembolic event,¹ with venous thrombosis the third most common cardiovascular disease after ischemic heart disease and stroke. Venous thrombosis affects 1 to 2 in 1000 individuals every year and is associated with life-threatening conditions such as pulmonary embolism (PE).¹ Though less clearly delineated, hypercoagulability is also believed to play a role in the pathogenesis of arterial thrombosis.²

Conditions associated with an increased risk of venous thrombosis can be either inherited or acquired (Tables 1 and 2). One or more predisposing factors are identifiable in 80% of individuals with a first episode of thrombosis, and an inherited cause of thrombophilia can be identified in approximately 30% to 35% of individuals with a first thrombotic episode.³ Manifestations include deep vein thrombosis (DVT) of the lower limbs, PE, superficial thrombophlebitis, mesenteric or cerebral vein thrombosis, fetal loss (spontaneous abortion or stillbirth), preeclampsia, and neonatal purpura fulminans.

Individuals at high risk for venous thrombosis include those with a personal or family history of thrombosis, inherited coagulation disorders, homocystinuria, paroxysmal nocturnal hemoglobinuria, essential thrombocythemia, polycythemia vera, recurrent spontaneous abortion and stillbirth, and malignancy. Additional risk factors include surgery, trauma, physical inactivity (bed confinement or paralysis), warfarin induced skin necrosis, diabetes, hyperlipidemia, vasculitis, thrombocytopenia, sepsis, congestive heart failure, and use of purified prothrombin complex concentrates. Other factors that may be associated with increased thrombotic risk include plasminogen deficiency and elevations of plasminogen activator inhibitor-1, lipoprotein (a), D-dimer, and thrombin-activatable fibrinolysis inhibitor. 

The risk of thrombosis increases with the number of defects or risk factors present; ie, individuals with multiple conditions associated with thrombosis are at greater risk than those with only one condition.¹ Risk factors for venous thrombophilia are generally not associated with risk of arterial thrombosis, with the exception of hyperlipidemia, antiphospholipid syndrome (Appendix 1,) elevated homocysteine, protein S deficiency, and dysfibrinogenemia.²

The identification of thrombotic risk factors and diagnosis of thrombophilia contributes to patient management in multiple ways (Table 3). Such diagnosis is based on personal and family history of thrombosis (especially during adolescence and young adult years), clinical manifestations, and laboratory testing.

Clear guidelines how to best manage individuals with a family or personal history of documented risk factors and who have not experienced a thrombotic episode have not been established. The decision for prophylactic therapy should be based on an individual‘s clinical history.¹² Screening general populations for inherited disorders associated with venous thrombosis is not recommended; however, the clinical utility of global screening assays in high-risk populations is being evaluated.

Patients with acute thrombosis are treated with intravenous heparin or oral anticoagulants such as warfarin (Coumadin^®′). Prophylactic treatment is provided to diagnosed patients when in high-risk situations, eg, surgery, prolonged immobilization, pregnancy and puerperium. Lifelong prophylactic therapy may be considered for those with recurrent thrombotic episodes, high-risk disorders, or with multiple-risk factors and may include plasma transfusions (eg, antithrombin concentrates), oral anticoagulants, low dose aspirin, and heparin.¹² Heparin is of limited benefit post thrombosis in patients with antithrombin deficiency, however, and heparin selection for pregnant women should be individualized due to risk of bone fracture.¹³ Low molecular weight heparin (LMWH) may be a better option for those at risk of osteoporosis since LMWH does not cause bone thinning. Individuals with hyperhomocysteinemia may be treated with vitamin supplementation (folic acid, cobalamin, pyridoxine).

• Symptomatic individuals

• Individuals with a personal or family history of thrombosis or thrombophilia-associated mutations

• High-risk individuals predisposed by surgery, trauma, immobility, pregnancy, oral contraceptives, etc.

Note: high-risk pregnant women include those with a personal or family history of thrombosis, previous neural tube defect affected fetus, recurrent spontaneous abortions, severe early onset preeclampsia, cesarean section, obesity, advanced maternal age, higher parity, and prolonged immobilization.¹³

Tests available to assist in diagnosis and management of thrombophilia disorders are listed in Appendix 2. Additionally, Quest Diagnostics offers panels that include multiple tests, thereby simplifying the test ordering process. Refer to the Quest Diagnostics Directory of Services for information on these panels, which are typically named according to the medical condition.

Diagnosis

A venous thrombosis laboratory work-up for high-risk or symptomatic individuals begins with a personal and family history. Test selection may vary for each individual based on his/her history as well as a particular defect’s prevalence in specific populations. For example, venous thrombosis in a pediatric patient suggests the likelihood of an inherited disorder; in an individual with SLE, antiphospholipid syndrome should be considered; and in an older individual, malignancy. Testing for multiple etiologies is recommended since venous thrombosis is a polyfactorial disorder, and presence of multiple etiologies increases the risk for thrombosis.^14,15 Generally accepted testing guidelines suggest the use of first and second line testing in the thrombophilia diagnosis (Figure).^14,17

First line testing for an individual with venous thrombosis typically includes a CBC with smear and APTT; activated protein C resistance (APCR); functional (activity) assays for antithrombin, proteins C and S, and factor VIII; prothrombin 20210G>A mutation detection; homocysteine; and anticardiolipin and antiphospholipid antibodies (see Appendix 1). APCR and prothrombin 20210G>A mutation detection need not be performed initially in non-Caucasian individuals since these disorders are primarily observed in Caucasians. Likewise, if a first thrombotic event occurs after the age of 50, testing for protein C, S, and antithrombin deficiency may be postponed as hypercoagulability due to these disorders usually manifests as thrombosis earlier than the fifth decade. Testing for heparin induced thrombocytopenia should be considered for any individual who has received heparin within the 30 days preceding a thrombotic episode and has a decrease in platelet count to <100,000/μL or more than 50% of baseline. Additional testing directed toward diagnosis of other causes of acquired thrombophilia such as systemic lupus erythematosus, liver disease, nephrotic syndrome, polycythemia vera, chronic myelogenous leukemia, diabetes mellitus, Cushing’s syndrome, etc. may be indicated (see Table 2).

Positive functional assays can be confirmed by genetic testing in some cases or by demonstration of the abnormality in another family member. For example, a borderline or positive APCR test can be confirmed with factor V (Leiden) mutation analysis. Such analysis differentiates homozygous and heterozygous states, providing additional prognostic information. Factor V HR2 allele mutation analysis provides even more prognostic information in factor V (Leiden) carriers. Homocysteine elevations may be due to an acquired nutritional deficiency (vitamin B₁₂, B₆, or folate) or a methylenetetrahydrofolate reductase (MTHFR) mutation. Acquired causes for antithrombin, protein C, and protein S deficiencies can be ruled out by liver function testing, a disseminated intravascular coagulation screen (D-dimer, fibrin degradation product, PT, APTT, fibrinogen, platelet count), and a proteinuria test (urine albumin).¹⁸ Decreased antithrombin and protein C and S activities (function) can be further characterized as a deficiency (type I or III) or dysproteinemia (type II) by using antigenic assays; however, such characterization will not affect treatment decisions.

If all of the aforementioned testing is negative, the patient may have a rare disorder that can be identified by testing for factors IX and XI, lipoprotein (a) [Lp(a)], plasminogen activity (function), plasminogen activator inhibitor-1 (PAI-1), and tissue plasminogen activator (TPA); evaluation for dysfibrinogenemia may also be helpful (Figure). Testing for rare disorders is only recommended for individuals with a strong personal and family history of thrombosis and negative first line tests or in whom clinical suspicion is high. Since all thrombophilia etiologies are not yet known, it is possible for all of these tests to be negative.

Management
Since individuals with variations in the CYP2C9 and VKORC1 genes may require lower warfarin doses, mutation analysis should be performed to assist in selecting the initial dosage and to prevent over anticoagulating the patient. Warfarin therapy can be monitored using the prothrombin time test, reported as INR, except in 1) some patients with a strong lupus anticoagulant and 2) patients with direct thrombin inhibitors. For these patients, monitoring with chromogenic factor X is preferred.

When injectible anticoagulants are used, patients can be monitored using an Xa inhibition assay. Quest Diagnostics offers 3 such tests, 1 for each class of injectibles (LMWH, unfractionated heparin, or synthetic LMWH). See Appendix 2 under Heparin and Fondaparinux for test details.

Acute thrombosis, anticoagulant therapy, drug therapy, and certain medical conditions can affect the results and interpretation of tests used to diagnose causes of thrombophilia (Tables 4 and 5). Additional interpretive information, specific to each test, is provided below.

Activated Partial Thromboplastin Time (APTT)
The APTT will be prolonged if there is deficiency or inhibition of factors of the intrinsic pathway including high molecular weight kininogen (HMWK), prekallikrein, factors V, VIII, IX, X, XI and XII, prothrombin, and fibrinogen. Prolongation is also seen in individuals with lupus anticoagulant.

Activated Protein C Resistance (APCR)
A decreased ratio of dRVVT clotting times obtained with and without activation of endogenous protein C is suggestive of an activated protein C (APC) inhibitor, a factor V (Leiden) mutation, and increased risk of deep vein thrombosis. Assay sensitivity and specificity approach 100%, even in the presence of anticoagulants and heparin (≤1 IU/mL plasma).^19,20 In <5% of cases, an APC inhibitor is found without a corresponding factor V (Leiden) mutation, perhaps indicative of an unknown mutation. Such cases are also associated with increased venous thrombosis risk.²¹

Annexin V
These antibodies have been associated with recurrent pregnancy loss, a clinical feature of antiphospholipid syndrome (see Appendix 1).

Antithrombin
Decreased levels of antithrombin are associated with an increased risk of both arterial and venous thrombosis and are seen in individuals with hereditary antithrombin deficiency, nephrotic syndrome, colitis, liver disease, active thrombosis, disseminated intravascular coagulation (DIC), those receiving l-asparaginase therapy or oral contraceptives, and individuals who are pregnant or have undergone surgery. Levels are also decreased in individuals receiving heparin. Levels in neonates are approximately half of the adult level, which is reached by 6 months of age. Low levels in both the activity and antigen assays indicate type I deficiency, whereas low activity levels in the presence of normal antigen levels indicate type II deficiency (dysproteinemia). Increased levels may be due to oral anticoagulants or heparin cofactor II.

C4 Binding Protein
Approximately 65% of protein S circulates in plasma bound to C4 binding protein. Increased levels of C4 binding protein may cause decreased levels of free protein S, and subsequent increased risk of thrombosis, and are associated with inflammation, pregnancy, diabetes mellitus, SLE, AIDS, allograft rejection, estrogen and progesterone administration, and smoking.

Cardiolipin Antibodies
Anticardiolipin antibodies of the IgA, IgG, and IgM isotype are associated with the antiphospholipid syndrome and, when >40 GPL units, increase the risk for venous thrombosis 5- to 8-fold. IgG antibodies appear to be more predictive of disease activity, while IgM antibody occurs more often in drug-induced disorders and infectious disease (eg, syphilis). Higher antibody titers are generally correlated with greater thrombotic risk (see Appendix 1).

CD55 and CD59 Expression, Red Cells and Granulocytes
Red cells and granulocytes deficient in CD55 and CD59 expression are associated with paroxysmal nocturnal hemoglobinuria (PNH); see Table 6. Individuals with PNH are at markedly increased risk of thrombosis, particularly of the intra-abdominal and cerebral veins; however, patients with <50% deficient granulocytes have a low risk of venous thrombosis. Anticoagulant therapy should be considered for patients with >50% deficient granulocytes.²²

Cytochrome P450 2C9 and VKORC1 Mutation Analysis
The cytochrome P450 enzyme CYP2C9 participates in the metabolism of a number of important drugs, including warfarin. Individuals carrying variants in the CYP2C9 gene (CYP2C9*2, CYP2C9*3, CYP2C9*5, and CYP2C9*6) have reduced metabolism of warfarin, and those with 2 copies of variant alleles are at high risk of life-threatening side effects. Presence of the -1639G>A mutation in the VKORC1 gene leads to a decrease in the level of vitamin K-dependent clotting factors and increased sensitivity to warfarin. VKORC1 and CYP2C9 polymorphisms, together with clinical factors, account for 50% to 60% of the variability in an individual’s response to warfarin^23-25; individuals with these polymorphisms may require lower warfarin doses.

D-Dimer
Elevated levels are associated with myocardial infarction, deep vein thrombosis, pulmonary embolism, DIC and other coagulation disorders, surgery, trauma, sickle cell disease, liver disease, severe infection, sepsis, inflammation, malignancy, obstetric complications, and hyperfibrinolysis. When clinical probability is low, a negative result (normal level) essentially rules out DVT.²⁶

dRVVT Screen with Reflex to dRVVT Confirm and dRVVT 1:1 Mix
This test evaluates the activity of factors II, V, and X (common clotting pathway). A confirmed dRVVT test result (ie, increased ratio) is indicative of a lupus anticoagulant and/or phospholipid antibody since excess phospholipid shortens (overrides) the prolonged dRVVT. A falsely prolonged dRVVT test may occur when heparin is >1.0 IU/mL. A false-negative dRVVT test may be due to platelet contamination of the plasma. Samples with moderate or severe icterus or lipemia are contraindicated.

Factor V HR2 Allele DNA Mutation Analysis
The HR2 allele is associated with APCR and increased risk of venous thrombosis in individuals also heterozygous for the factor V (Leiden) mutation. Such co-inheritance increases the risk of venous thromboembolism 3- to 4-fold when compared with factor V (Leiden) alone. An individual heterozygous positive for the HR2 allele and negative for factor V (Leiden) is not at increased risk of thrombosis compared to factor V (Leiden) alone. However, homozygosity for factor V HR2 is associated with increased risk of thrombosis even in the absence of a factor V (Leiden) mutation.

Factor V (Leiden) Mutation Analysis
The 1691G>A factor V Leiden mutation results in the laboratory finding of APCR. Factor V (Leiden) confers approximately a 7-fold increase in venous thromboembolic events in heterozygous individuals and an 80-fold increase in homozygous subjects.²⁷ When a heterozygous mutation is coupled with oral contraceptive use, the risk increases synergistically to 30-fold.²⁸ The mutation is also associated with arterial thrombosis (especially in smokers), complications of pregnancy (including fetal loss),²⁹ and increased levels of factor VIII. Although this test is highly specific, identification of a mutation may occur in the absence of APCR in rare cases. A negative result does not rule out APCR or an increased risk of venous thrombosis.

Factor VIII
Factor VIII is an acute phase reactant and increased levels are found during periods of stress, postoperatively, and in inflammatory conditions. Elevated levels are also found at birth and during pregnancy. Increased levels are associated with increased risk for venous thrombosis,³⁰ whereas decreased levels are associated with hemophilia A. A factor VIII activity:fibrinogen ratio >0.75 is considered diagnostic of factor VIII excess.

Fibrin Monomer
The presence of soluble fibrin monomer complexes in plasma is diagnostic of DIC.

Fibrinogen
Increased levels are associated with acute phase reactions, pregnancy, and increased risk of thrombosis. Low fibrinogen activity levels are associated with afibrinogenemia, hypofibrinogenemia, or dysfibrinogenemia (which may be associated with thrombophilia in rare instances), as well as with DIC, systemic fibrinolysis, pancreatitis, severe hepatic dysfunction, and l-asparaginase or valproate treatment. Individuals with afibrinogenemia or hypofibrinogenemia will have decreased activity and antigen levels. Individuals with dysfibrinogenemia will typically have decreased activity levels and normal or decreased antigen levels

Fibrinogen Degradation Products (FDP)
FDP result from the breakdown of fibrinogen, as well as fibrin, by plasmin. Normally, the fibrinolytic process is localized to fibrin, however, during conditions such as DIC, fibrinolysis spreads and becomes systemic. Elevated levels of FDP are seen in many clinical states (eg, DVT and PE); thus measurement of FDP is useful for their diagnosis. Persistent elevations indicate that abnormal fibrinolysis and fibringenolysis are occurring.

Fondaparinux Sodium (Xa Inhibition)

Fondaparinux is a synthetic pentasaccharide administered subcutaneously and used to prevent or treat thromboembolic conditions. Measurement is used to monitor therapeutic levels. The therapeutic range is 1.20-1.26 μg/mL, and the prophylactic range is 0.39-0.50 μg/mL. These ranges are applicable to samples collected approximately 3 hours after administration of the drug.

β₂-Glycoprotein I Antibodies
β₂-Glycoprotein I antibodies of the IgA, IgG, and IgM isotype are associated with the antiphospholipid syndrome, and their presence is more specific but less sensitive than cardiolipin antibodies for the diagnosis of antiphospholipid syndrome. Individuals who are positive for cardiolipin antibodies and negative for β₂-glycoprotein I antibodies are more likely to have an infection (varicella, rubella, adenovirus, HIV) or drug exposure (amoxicillin, chlorpromazine, hydralazine) than antiphospholipid syndrome (Appendix 1).

Heparin Anti-Xa (Low Molecular Weight Heparin)
LMWH are prepared by the chemical or enzymatic degradation of unfractionated heparin, and are used in the prevention and treatment of thromboembolic conditions. Measurement of LMWH in plasma is used to monitor therapeutic levels. The therapeutic and prophylactic ranges for samples collected 4 hours after subcutaneous administration are shown in Table 7. This assay was validated using enoxaprin; thus it may not be appropriate for optimal evaluation of patients receiving other LMWH.

Heparin Anti-Xa (Unfractionated Heparin)
Unfractionated heparin is used for the prevention and treatment of thromboembolic conditions and measurement is used to monitor therapeutic levels. When administered as an intravenous infusion, the therapeutic range is 0.30 to 0.70 IU/mL

Homocysteine
Levels are increased in the following: cardiovascular disease, vitamin B₁₂ and folate deficiencies, chronic renal disease, homocystinuria, hypothyroidism, selected malignancies, individuals whose diet is rich in methionine (high meat intake), cigarette smokers, and in individuals treated with corticosteroids, methotrexate, cyclosporin, vitamin B₆ antagonists (isoniazid, azauridine, penicillamine, procarbazine), anticonvulsants (phenytoin, carbamazepine), and S-adenosyl-methionine. When coupled with the factor V (Leiden) mutation, venous thrombosis risk increases synergistically.³¹ Falsely increased levels may occur if serum or plasma is not separated from the red cells within 1 hour of collection.

Homocysteine is decreased in pregnancy (except in some women carrying a fetus with a neural tube defect), individuals less than 15 years of age, and individuals taking oral contraceptives or hormone replacement therapy.

Human Platelet Antigen 1 (HPA-1) Genotype
The HPA-1b platelet antigen polymorphism is associated with increase platelet thrombogenecity, neonatal alloimmune thrombocytopenia, and post-transfusion purpura.

Lipoprotein (a) [Lp(a)]
Increased levels of Lp(a) are observed in patients with coronary artery disease, stroke, cerebrovascular and peripheral vascular disease, and venous thrombosis. Substantial increases are secondarily (not genetically related) observed in nephrotic syndrome and end-stage renal disease. Decreased Lp(a) levels may be seen in several rare disorders (lecithin:cholesterol acyltransferase [LCAT] deficiency, lipoprotein lipase [LPL] deficiency, liver disease). Normal levels in the African American population may be 2 to 3 times the values in Caucasian and Asian populations. Native Americans and Mexican Americans have lower normal levels (no lower than one half) relative to the Caucasian and Asian populations.

Methylenetetrahydrofolate Reductase (MTHFR) DNA Mutation Analysis
A homozygous MTHFR mutation (677C>T) may be associated with hyperhomocysteinemia, an increased risk for arterial and venous thrombosis, and an increased risk for obstetrical complications (eg, preeclampsia, abruptio placenta, fetal growth retardation, and stillbirth).²⁹ Heterozygosity for this mutation, in the absence of vitamin deficiency, usually is associated with normal plasma homocysteine levels.

Mixing/Correction Study
Test results are consistent with an intrinsic factor deficiency when a prolonged APTT is normalized after mixing patient plasma with normal plasma and the normalized result does not reverse after incubation of the mixed sample. A specific factor inhibitor, lupus inhibitor, fibrinolysis, or fibrinogenolysis is suggested when 1) the PT is normal, and the APTT is prolonged initially, normalizes after mixing, and reverses to prolonged after incubation of the mixed sample; 2) the PT is normal, and the APTT remains prolonged after mixing studies; 3) a prolonged PT is corrected in the mixing study, and a prolonged APTT remains prolonged; 4) a prolonged PT remains prolonged, and a prolonged APTT normalizes after mixing, and reverses to prolonged after incubation. A prolonged PT that is corrected in the mixing study, along with a normal APTT, suggests a factor VII deficiency. Test results are consistent with a single or multiple deficiency of factors II, V, or X (common pathway) when a prolonged PT is normalized after mixing studies and when a prolonged APTT normalizes after mixing studies and remains normalized after incubation.

Phosphatidylserine, Phosphatidylinositol, Phosphatidylcholine, Phosphatidylethanolamine, Phosphatidylglycerol, and Phosphatidic Acid Antibodies
These antibodies may be associated with thrombosis and/or recurrent pregnancy loss, clinical features of antiphospholipid syndrome (see Appendix 1).

Plasminogen Activator Inhibitor-1 (PAI-1)
Increased levels of PAI-1 antigen and PAI-1 activity are associated with decreased fibrinolytic activity and increased risk for venous thrombosis and coronary artery disease. Interpretation of increased levels is confounded by circadian variation (morning values being about 2-fold higher than afternoon values), increases associated with the acute phase response (eg, following myocardial infarction, major surgery, severe trauma, or sepsis), as well as increases associated with normal pregnancy and disorders such as endotoxemia, liver disease, obesity, hyperinsulinemia, hypertriglyceridemia, and malignancy. Severely decreased or undetectable levels may be associated with bleeding problems. The antigenic, but not the activity test can help distinguish between a constitutional deficiency of PAI-1 and a dysproteinemia.

Plasminogen Activator Inhibitor-1 (PAI-1) 4G/5G Polymorphism
The 4G allele is associated with increased PAI-1 antigen and activity levels. Similar to PAI-1 antigen and activity levels, data regarding utility of the 4G/5G polymorphism in predicting venous thrombosis is conflicting.³² It may be more useful when co-inherited with another thrombophilia marker. When co-inherited, the 4G allele may further increase the risk of thrombophilia. For example, Visanji et al found the risk for venous thrombosis increased approximately 2-fold in patients with at least 1 copy of the 4G allele (4G/4G or 4G/5G genotypes) relative to that in patients with the 5G/5G genotype.³³ All patients were heterozygous for factor V (Leiden) mutation and had experienced at least 1 venous thromboembolic event. Furthermore, Zoller et al reported an approximate 4-fold increase in the risk of pulmonary embolism among subjects with hereditary protein S deficiency who were homozygous for the 4G allele.³⁴

Plasminogen
Decreased levels are associated with liver disease, DIC, thrombolytic agents, primary fibrinolysis, tissue plasminogen activator and, rarely, with venous thrombosis and pulmonary embolism (homozygous state only). Increased levels are associated with trauma, infection, acute myocardial infarction, surgery, and chronic inflammation. A functional assay is usually the preferred assay because the presence of a normal amount of antigen does not exclude a qualitative defect of the protein. The antigen level is most often obtained to assess a quantitative abnormality of the protein.

Protein C
Decreases are associated with venous thrombosis, recurrent superficial thrombophlebitis, neonatal purpura fulminans, arterial thrombosis (rarely), oral anticoagulant-induced skin necrosis, DIC, infection, acute illness such as the flu or a gastrointestinal disorder, malignancy, liver disease, vitamin K deficiency, surgery, and l-asparaginase therapy. Falsely low values may be obtained in individuals on oral anticoagulant therapy and those who are APC resistant. Heparin levels up to 1 IU/mL do not interfere with test results. Protein C levels are significantly decreased in neonates; adult levels are reached only after 10 years of age. Low levels in both activity and antigen assays are suggestive of type I deficiency, whereas low activity levels in the presence of normal antigen levels indicate type II deficiency (dysproteinemia). Increases are associated with oral contraceptives, and pregnancy. Although not affected by increased factor VIII or acute phase reactions, overall specificity for inherited deficiency is low, and positive results (ie, low levels) should be confirmed by demonstration of a deficiency in another family member.

Protein S
Decreases are associated with increased risk for venous, and possibly arterial, thrombosis, oral anticoagulant-induced skin necrosis, neonatal purpura fulminans, DIC, acute phase reactions, oral anticoagulants, APC resistance, vitamin K deficiency, liver disease, surgery, l-asparaginase therapy, oral contraceptives, estrogen replacement therapy, pregnancy, nephrotic syndrome, infections (HIV, varicella), Crohn‘s disease, and ulcerative colitis. Levels are significantly decreased in neonates; however, adult levels are reached by 6 months of age. Low levels in both activity and antigen (free and total) assays are suggestive of type I deficiency, whereas low activity in the presence of normal total antigen levels indicate type II deficiency (dysproteinemia). Type III deficiency is characterized by low levels in the activity and free antigen assays, but normal levels in the total antigen assay. Increases may be observed in lipemic samples. Although protein S levels are not affected by heparin (up to 1 IU/mL) or factor VIII (up to 250%), overall specificity of protein S measurement for the diagnosis of inherited deficiency is low, and positive results (ie, low levels) should be confirmed by demonstration of a deficiency in another family member.

Prothrombin (Factor II) 20210G>A Mutation
This mutation is associated with increased prothrombin levels, increased risk for venous thrombosis,¹¹ increased risk for obstetric complications (eg, preeclampsia, abruptio placenta, fetal growth retardation, and stillbirth),²⁹ and, possibly, premature coronary heart disease. Venous thrombosis risk increases synergistically in the presence of oral contraceptive use.³⁵ The combination may also lead to cerebral sinus and spinal vein thromboses.

Prothrombin Fragment 1.2
Prothrombin fragment 1.2 is the amino terminus fragment of prothrombin released when prothrombin is converted to thrombin. Elevated levels are associated with an increased risk of thrombosis and found in patients with trauma, eclampsia, pre-eclampsia, DIC, DVT, and PE. Levels are also increased in individuals with antithrombin deficiency.

Prothrombin Time (PT)

Prolonged PT results suggest a potential bleeding disorder that may be caused by a deficiency in factor II, V, VII, or X. Prolonged results are also associated with very low fibrinogen levels, factor-specific inhibitors, or a lupus anticoagulant (minimal increase). When patients taking anticoagulants such as warfarin have a prolonged result, they are at risk of a bleeding episode, and a dose adjustment should be considered. Results are interpreted based on the international normalized ratio (INR).

PTT-LA with Reflex to Hexagonal Phase Confirm
Lupus anticoagulants are non-specific antibodies that extend the clotting time of phospholipid-dependent clotting assays, and lupus anticoagulant antibodies bind to hexagonal phase phospholipids. A reduction of the APTT by more than 8 seconds after the addition of hexagonal phase phospholipid is considered confirmation of the presence of a lupus anticoagulant and an increased risk of thrombosis. The sensitivity and specificity of this test are 96% and >95%, respectively. False-positives may be seen with specific factor inhibitors (factors VIII and V) and direct thrombin inhibitors. While false-negatives are rare, if clinical suspicion of LA is high, dRVVT with confirm is suggested.

Reptilase Clotting Time
Unlike thrombin, reptilase is not affected by the presence of heparin or hirudin; thus, a prolonged thrombin time in an individual with a normal reptilase time is consistent with contamination or the presence of heparin. Reptilase clotting time is also prolonged in individuals with a-, hypo-, and dysfibrinogenemias.

Serotonin Release Assay (SRA)
A value >20% is considered positive and strongly suggests heparin induced thrombocytopenia.

Thrombin-Antithrombin (TAT) Complex
Elevated TAT complex is a risk factor for thrombosis and found in individuals with DIC, malignancies, and those receiving heparin and fibrinolysis therapy.

Thrombin Clotting Time
Prolonged clotting times may indicate abnormal fibrinogen levels (elevated or decreased), dysfibrinogenemia, the presence of heparin, hirudin, paraproteins, uremia, or increased levels of fibrin degradation products. Normalization of clotting time after mixing indicates hypo- or dysfibrinogenemia, whereas continued prolongation indicates the presence of heparin, hirudin, paraproteins, uremia, or increased levels of fibrin degradation products.

Tissue Plasminogen Activator (TPA)
TPA converts plasminogen to plasmin, which in turn degrades fibrin to soluble degradation products. TPA is inhibited by plasminogen activator inhibitor-1. Elevated TPA levels are associated with an increased risk of atherosclerosis, myocardial infarction, stroke, and recurrent venous thrombosis.

von Willebrand Factor Protease (ADAMTS-13) Activity with Reflex to Protease Inhibitor
von Willebrand factor is released into circulation as high molecular weight multimeric forms that are broken down into smaller, less active multimers by von Willebrand factor cleaving protease (ADAMTS-13). The persistence of high weight multimers is associated with platelet aggregates and thrombi. Deficiency of von Willebrand factor cleaving protease is associated with thrombotic thrombocytopenic purpura.