Hypercoagulable States - Thrombophilia

Diagnosis

Indications for Testing

  • Patient with new or recurrent venous thromboembolism (VTE) without obvious acquired risk factors (see Differential Diagnosis, below)
    • Abnormalities may be identified in a significant number of patients; however, identification of an abnormality may not predict risk of recurrence or alter therapeutic plan
    • Use when the results will impact management of the patient or patient family members
  • Situations where testing should be considered (College of American Pathologists [CAP], American College of Medical Genetics [ACMG])
    • Idiopathic thrombosis in patient ≤50 years of age
    • Recurrent thrombosis
    • Unusual sites of thrombosis (without risk factor)
    • First-degree relatives with thromboses
    • Thrombotic event during pregnancy or while taking oral contraceptives

Laboratory Testing

  • Based on family and patient history – nongenetic tests may be altered by anticoagulant therapy or acute phase reaction due to clot

  • Consider acquired disorders such as antiphospholipid syndrome
  • If testing pursued, consider the following combination (ACMG)
    • Activated protein C resistance (with or without reflex to factor V Leiden [FVL] mutation); factor V R2 A4070G mutation
    • Prothrombin mutation
    • Antithrombin (AT) activity
    • Protein C activity
    • Free protein S
    • Testing for less-common disorders may be considered if results are uninformative and additional testing is considered necessary
  • The following tests are not recommended
    • Factor VIII activity (testing other factor activities such as FVIII and FIX is controversial and not currently recommended)
    • Factor XIII (F13A1) V34L variant – presence of variant associated with decreased/reduced risk for deep vein thrombosis, myocardial infarction and coronary artery disease in Caucasians
  • Before making a definitive diagnosis of an inherited thrombophilia, consider repeating abnormal functional or antigenic testing
    • Low results can be obtained due to patient condition/biologic variability, medications, and assay variability or interference
    • Normal ranges vary by age and gender and must be considered when interpreting results

Differential Diagnosis

  • Acquired thrombophilia is more common than inherited thrombophilia and its causes and should be considered when evaluating patients with thrombosis

Screening

  • Population testing is not recommended for unselected patients

Clinical Background

Hereditary thrombophilia is a genetically determined increased risk for thrombosis and thromboembolism. Hypercoagulable states may also be acquired.

Epidemiology

  • Adults
    • Incidence – 30-500/10,000
      • Inherited thrombophilic disorders

        Inherited Thrombophilic Disorders

        Disorder

        Prevalence in Normals (%)

        Frequency in Patients with VTE+ (%)

        Relative Risk of First Episode of DVT++

        Factor V Leiden (heterozygous)

        0.05-4.8*

        18.8

        7

        Factor V Leiden (homozygous)

        0.02

        1.5

        80

        Factor V with R2 mutation

        0.06-0.12

        10.0

        7 (heterozygote)

        10 (compound heterozygote)

        Prothrombin G20210A allele

        0.06-2.7*

        7.1

        2.8

        Protein C deficiency

        0.2-0.4

        3.7

        6.5

        Protein S deficiency

        0.2-0.4

        2.3

        5.0

        Antithrombin deficiency

        0.02-0.2

        1.9

        20

        Dysfibrinogenemia

        <0.01

        0.8

        Unknown

        Hyperhomocysteinemia**

        5-7

        10

        2.95

        Elevated factor VIII level

        11

        25

        4.8

        Elevated factor IX level

        10

        20

        2.8

        Elevated factor XI level

        10

        19

        2.2

        Elevated lipoprotein (a) level

        7

        20

        3.2

        Elevated thrombin-activatable fibrinolysis inhibitor (TAFI)

        9

        14

        1.7

        *Percent lowest in Asian or African descent; highest in Caucasian descent
        **>18.5 µmol/L
        +VTE = venous thromboembolism
        ++DVT = deep vein thrombosis

Pediatrics

  • Incidence – 0.05-14/100,000
  • Age – peak in neonates and infants <1 year
    • Second peak in puberty
  • Sex – M<F

Etiologies

  • Most common thrombophilias
    • Factor V Leiden  
    • Prothrombin G20210A
  • Less common thrombophilias
    • Increased clotting factors
      • Elevated factor VIII (FVIII) levels are often found in patients with venous thrombosis, but routine testing is controversial
    • Protein C deficiency
    • Protein S deficiency
    • Hyperhomocysteinemia (acquired or inherited)
    • Antithrombin deficiency
    • Impaired clot lysis (dysfibrinogenemia, abnormal fibrinolysis)
  • Antiphospholipid syndrome is an acquired thrombophilic state

Factor V Leiden

Genetics and Pathophysiology

  • Factor V Leiden (FVL) mutation of the F5 gene is the most common inherited thrombophilia
    • Accounts for more than 90% of patients with activated protein C resistance (APC-R)
      • During normal hemostasis, APC limits clot formation by proteolytic inactivation of factors Va and VIIIa
      • FVL prevents inactivation of factor Va by APC at the normal rate, increasing the risk for thrombosis
    • Functional tests for APC-R are generally used to screen for FVL
      • DNA tests are used to confirm positive screening tests and to differentiate between heterozygotes and homozygotes
  • Autosomal dominant inheritance
    • Heterozygotes have a five- to tenfold increased risk
    • Homozygotes have a 50- to 100-fold increased risk

Clinical Presentation

  • Venous thromboembolism (VTE) is the most common type of thrombotic event
    • Recurrent VTE (pulmonary embolism, DVT) is uncommon in heterozygotes unless additional risk factors are present
    • Increased risk of recurrent VTE in homozygotes
  • Recurrent miscarriage in the second trimester of pregnancy

Additional Risk Factors

  • Presence of F5 R2 (A4070G) mutation with FVL increases risk of thrombotic event tenfold
  • Patients with FVL mutation and recurrent episodes of thrombosis often have more than one genetic risk factor (eg, concomitant prothrombin G20210A mutation of F2, protein C deficiency, homocystinemia)
  • Acquired factors such as pregnancy, oral contraceptives, hormone replacement therapy, and immobilization increase the risk

Prothrombin G20210A

Genetics and Pathophysiology

  • Prothrombin G20210A mutation of the F2 gene is the second most common inherited thrombophilia
    • Results in elevated levels of plasma prothrombin leading to hypercoagulability (gain of function)
    • Detected using DNA tests
      • Factor II (prothrombin) activity testing may not identify the disease and should not be used for diagnosis
  • Autosomal dominant inheritance
    • Variable penetrance
      • Many patients who are either heterozygous or homozygous for G20210A do not experience VTE
    • Heterozygosity causes a two- to fourfold increase in thrombotic risk
    • Homozygosity is rare and increases thrombotic risk above that seen in G20210A heterozygotes

Clinical Presentation

  • VTE
  • Pregnancy complications – preeclampsia, placental abruption

Additional Risk Factors

  • Combined heterozygosity for both prothrombin G20210A and FVL mutations leads to thrombophilia with earlier onset, higher rate of recurrence, and more severe thrombotic events than either mutation alone
  • Increased risk of thrombosis associated with oral contraceptive use and pregnancy

Protein C Deficiency

Genetics and Pathophysiology

  • Protein C is a vitamin K-dependent plasma anticoagulant activated to APC by thrombin-thrombomodulin, which then inactivates factors Va and VIIIa
    • Inherited protein C deficiency is uncommon
      • Two forms
        • Type I – quantitative
        • Type II – qualitative
      • Autosomal dominant inheritance
        • Highly variable phenotypic expression
      • Functional assays preferred for diagnosis (rather than antigenic assays)
        • Protein C levels vary with age
    • Protein C levels are decreased in acute thrombotic states, disseminated intravascular coagulation (DIC), liver disease, malnutrition (vitamin K deficiency), and with warfarin therapy
    • Increased protein C levels may be seen in diabetes, nephrotic syndrome, pregnancy, and with oral contraceptive use
      • Elevated FVIII levels may result in falsely decreased values in some functional assays
      • Heparin and direct thrombin inhibitors may result in falsely elevated values in some functional assays

Clinical Presentation

  • Additional risk factors likely necessary to provoke thrombosis
  • VTE in heterozygotes
  • Neonatal purpura fulminans (DIC) in homozygous infants – widespread thromboses
  • Warfarin-induced skin necrosis is rarely seen

Protein S Deficiency

Genetics and Pathophysiology

  • Protein S is a vitamin K-dependent plasma anticoagulant that acts as a cofactor for activated protein C and exists in two forms
    • Free protein S – 40% of the total; physiologically active
    • Bound protein S (attached to C4b-binding protein) – 60% of the total; no anticoagulant activity
  • Inherited protein S deficiency is uncommon
    • Three forms
      • Type I – quantitative
      • Type II – qualitative
      • Type III (also called type IIa) – quantitative with normal levels of total protein S
    • Autosomal dominant inheritance
    • Antigenic tests for free protein S preferred for diagnosis
      • Free protein S values are higher in males than in females
  • Protein S values are decreased in acute thrombotic states, nephrotic syndrome, inflammatory syndromes (due to increased C4b-binding protein), DIC, liver disease, malnutrition (vitamin K deficiency), pregnancy, and with estrogen and warfarin therapy
    • Elevated FVIII levels and/or APC resistance may result in falsely decreased values in some functional assays
    • Heparin and direct thrombin inhibitors may result in falsely elevated values in some functional assays

Clinical Presentation

  • Additional risk factors likely necessary to provoke thrombosis
  • VTE most common; arterial thrombosis may occur
  • Neonatal purpura fulminans (DIC) in homozygous infants
  • Warfarin-induced skin necrosis (rare)

Antithrombin Deficiency

Genetics and Pathophysiology

  • Antithrombin (AT) – plasma anticoagulant; inactivates thrombin, factor Xa, and other activated clotting factors
    • AT activity enhanced by heparin-like glycosaminoglycans on the endothelial surface and by pharmaceutical heparin
    • Synthesized in the liver
  • Two forms of inherited antithrombin deficiency
    • Type I – quantitative
    • Type II – qualitative
  • Autosomal dominant inheritance
    • Functional assays preferred for diagnosis
    • Homozygous state is embryonic lethal
  • Decreased AT occurs in acute thrombotic states, liver disease, DIC, nephrotic syndrome, and heparin therapy; mild decreases may be seen in pregnancy or with oral contraceptive use
  • Increased AT may occur with long-term warfarin therapy

Clinical Presentation

  • VTE
  • Recurrent thrombosis may occur even in the absence of additional risk factors
  • Some deficient patients are resistant to heparin therapy

Hyperhomocysteinemia

Acquired

  • Independent risk factor for thromboembolic events
  • Most patients with hyperhomocysteinemia have no genetic mutations or polymorphisms
  • Acquired hyperhomocysteinemia is the result of defective homocysteine metabolism and may be the result of  vitamin B6, B12, or folic acid deficiency
  • Thrombotic risk most closely associated with increased fasting plasma homocysteine levels regardless of underlying etiology
    • Plasma homocysteine testing is recommended over DNA-based tests

Inherited

  • Homocysteine metabolism defects caused by mutations of the MTHFR gene
    • c.665C>T (previously c.677C>T) and c.1286A>C (previously c.1298A>C) most common
      • c.665C>T results in a thermolabile variant of MTHFR
  • Autosomal recessive inheritance
  • Elevated plasma homocysteine levels have been associated with atherosclerotic disease, VTE, and arterial thrombosis
  • Inherited hyperhomocysteinemia rare
    • Clotting Cascade with an Emphasis on Inherited Thrombophilias

Indications for Laboratory Testing

  • Tests generally appear in the order most useful for common clinical situations
  • Click on number for test-specific information in the ARUP Laboratory Test Directory
Test Name and Number Recommended Use Limitations Follow Up
APC Resistance Profile with Reflex to Factor V Leiden 0030192
Method: Electromagnetic Mechanical Clot Detection/Polymerase Chain Reaction/Fluorescence Monitoring

Recommended test to detect activated protein C resistance and confirm presence of a factor V Leiden mutation

Initial screening for suspected thrombophilia

Profile includes thrombosis common etiologies, factor VIII activity, PTT, prothrombin G20210A mutation; reflex to factor V Leiden

APC resistance profile may be affected by heparin levels above 2 IU/mL, direct thrombin inhibitors, and low factor V activity levels (<50%)

Perform PCR testing as first-line test if these are present

APC resistance due to a cause other than a factor V mutation will not be detected

 
Thrombotic Risk, Inherited Etiologies (Uncommon) 0030177
Method: Electromagnetic Clot Detection/Microlatex Particle-Mediated Immunoassay/Chromogenic Assay

Acceptable panel to screen for uncommon inherited thrombophilias

Prior to ordering screen, screen for APC resistance

Panel includes PTT, free functional protein C, protein S antigen, antithrombin enzymatic activity

See individual components

See individual components

Protein C, Functional with Reflex to Protein C, Total and Protein S, Free with Reflex to Protein S, Total 2003386
Method: Electromagnetic Mechanical Clot Detection/Enzyme-Linked Immunosorbent Assay/Microlatex Particle-Mediated Immunoassay

Acceptable panel to detect and subtype protein C and S deficiencies

Do not order if the individual has been on warfarin therapy in the previous 2-4 weeks

   
Protein S, Free Antigen with Reflex to Protein S, Total Antigen 2002269
Method: Microlatex Particle-Mediated Immunoassay

Use to detect and subtype protein S deficiency

 Do not order if individual has been on warfarin therapy in the previous 2-4 weeks

Reflex pattern – if low protein S free antigen is detected, then protein S total antigen will be added

   
Thrombotic Risk (Acquired) Reflexive Panel 0030268
Method: Electromagnetic Clot Detection/Semi-Quantitative Enzyme-Linked Immunosorbent Assay/Immunoturbidimetry/Quantitative Enzymatic

Acceptable screening panel for acquired thrombophilia; not considered initial tests in evaluation of thrombophilia

Panel includes antiphospholipid antibodies, d-dimer and clotting time assays, and homocysteine

See individual components

Interpretation provided in test report

Thrombotic Risk, Inherited Etiologies (Most Common) with Reflex to Factor V Leiden 0030133
Method: Electromagnetic Clot Detection/Quantitative Enzymatic/Polymerase Chain Reaction/Fluorescence Monitoring

Acceptable panel to screen for common inherited thrombophilias

Panel includes APC resistance profile, prothrombin G20210A mutation, aPTT, FVIII activity, and homocysteine

Reflex pattern – if APC resistance is normal, then no further testing will be added; if APC resistance is abnormally low, then Factor V Leiden by PCR will be added

See individual components

See individual components

Additional Tests Available
 
Click the plus sign to expand the table of additional tests.
Test Name and NumberComments
APC Resistance Profile 0030127
Method: Electromagnetic Mechanical Clot Detection

Acceptable initial test to detect activated protein C resistance due to a factor V Leiden mutation

Preferred test is APC resistance profile with reflex to factor V Leiden;  however, factor V Leiden (F5) R506Q mutation is the preferred initial test for individuals with supratherapeutic concentrations of heparin, direct thrombin inhibitors, extreme factor V deficiency, or lupus anticoagulants with markedly prolonged baseline clotting times

Prothrombin (F2) G20210A Mutation 0056060
Method: Polymerase Chain Reaction/Fluorescence Monitoring

Order to detect prothrombin G20210A mutation

Antithrombin, Enzymatic (Activity) 0030010
Method: Chromogenic Assay

Recommended test to detect antithrombin (AT) deficiency

Antithrombin Panel 0030370
Method: Chromogenic Assay/Microlatex Particle-Mediated Immunoassay

Order to detect and subtype AT deficiency

Antithrombin, Antigen 0030015
Method: Microlatex Particle-Mediated Immunoassay

Not recommended as an initial test to detect AT deficiency

Use to determine subtype in AT-deficient individuals

Protein C and S Panel, Functional 0030182
Method: Electromagnetic Mechanical Clot Detection

Acceptable panel to detect protein C and S deficiencies

Preferred test for detecting protein S deficiency is protein S free antigen

Do not order if the individual has been on warfarin therapy in the previous 2-4 weeks

Protein C, Functional with Reflex to Protein C, Total Antigen 0030041
Method: Electromagnetic Mechanical Clot Detection/Enzyme-Linked Immunosorbent Assay

Acceptable test to detect and subtype protein C deficiency

Do not order if individual has been on warfarin therapy in the previous 2-4 weeks

Protein C and S Panel, Total, Antigen 0030116
Method: Microlatex Particle-Mediated Immunoassay

Not recommended

Protein S Free, Antigen 0098894
Method: Microlatex Particle-Mediated Immunoassay

Recommended test to detect protein S deficiency

Do not order if individual has been on warfarin therapy in the previous 2-4 weeks

Protein S, Functional 0030114
Method: Electromagnetic Mechanical Clot Detection

Acceptable test for detecting protein S deficiency

Preferred test is protein S free, antigen

Do not order if the individual has been on warfarin therapy in the previous 2-4 weeks

Protein S, Total Antigen 0030112
Method: Microlatex Particle-Mediated Immunoassay

Not recommended for detecting protein S deficiency; preferred test is protein S free antigen

Use to subtype deficiency in known protein S-deficient individuals in combination with protein S free antigen or protein S functional

Do not order if individual has been on warfarin therapy in the previous 2-4 weeks

Protein C, Functional 0030113
Method: Electromagnetic Mechanical Clot Detection

Recommended test to detect protein C deficiency

Do not order if individual has been on warfarin in the previous 2-4 weeks

Protein C, Total Antigen 0030111
Method: Enzyme Immunoassay

Not recommended for detecting protein C deficiency; preferred test is protein C functional

Use to subtype deficiency in known protein C-deficient individuals

Do not order if individual has been on warfarin therapy in the previous 2-4 weeks

Homocysteine, Total 0099869
Method: Quantitative Enzymatic

Not recommended for risk assessment of CVD or VTE

Acceptable screening test for disorders of methionine metabolism

Methylenetetrahydrofolate Reductase (MTHFR) 2 Mutations 0055655
Method: Polymerase Chain Reaction/Fluorescence Monitoring

Do not use for thrombophilia screening

Factor VIII, Activity 0030095
Method: Electromagnetic Mechanical Clot Detection

Order to diagnose hemophilia A, acquired factor VIII deficiency, or as part of a diagnostic workup for von Willebrand disease

Not recommended when screening for thrombophilia

Factor V, R2 Mutation 2001549
Method: Polymerase Chain Reaction/Restriction Enzyme Digestion/Gel Electrophoresis

Order to determine thrombotic risk in individuals known to be factor V Leiden heterozygotes

Factor XIII (F13A1) V34L Variant 2003220
Method: Polymerase Chain Reaction/Fluorescence Monitoring

Assess genetic risk for thrombosis

Thrombotic Risk, DNA Panel 0056200
Method: Polymerase Chain Reaction

Acceptable panel to detect two most common inherited thrombophilias

Includes Factor V Leiden (F5) R506Q, MTHFR 2 mutations (c.665C>T and c.1286A>C), and prothrombin (F2) G20210A mutations

Factor V Leiden (F5) R506Q Mutation 0097720
Method: Polymerase Chain Reaction/Fluorescence Monitoring

Order to detect factor V Leiden mutation