Hypercoagulable States - Thrombophilia

Diagnosis

Indications for Testing

• Patient with new or recurrent 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 change therapy
  • Test in cases where the results will impact management of the patient or patient family members
• Situations where testing should be considered (College of American Pathologists, 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
  • Thrombotic event while taking oral contraceptives

Laboratory Testing

• Test ordering based on family and patient history
• Consider acquired disorders such as
  • Antiphospholipid syndrome
  • Hyperhomocysteinemia (associated cardiovascular disease)
  • Cancer screening based on age and risk factors
• If testing indicated, consider the following (not known whether testing improves outcomes)
  • Activated protein C resistance (with or without reflex to FVL mutation); Factor V R2 A4070G mutation (Factor V Leiden)
  • Prothrombin mutation
  • Antithrombin activity
  • Protein C activity
  • Free protein S
  • Factor VIII activity 
  • Testing for less-common disorders is available if results are uninformative or additional testing is indicated
• 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
• If factor V Leiden mutation found, the ACMG recommends the following
  • Genetic testing – not affected by an acute phase response or anticoagulant therapy
    • Prothrombin mutation G20210A of F2
    • Factor V, R2 mutation of F5
  • Measurement of total plasma homocysteine concentration
  • Functional coagulation assays for antithrombin III, protein C and protein S deficiencies
    • May be altered by acute thrombotic states (acute phase response, consumption of factors and anticoagulant factors) and anticoagulant therapies
      • Delay testing 2-3 months after acute event
      • Discontinue oral anticoagulant therapy at least 2 weeks to 1 month before testing
      • Heparin and direct thrombin inhibitors interfere with many of the tests and should be discontinued prior to testing
        • Heparin interference in tests may be due to therapy with unfractionated or low molecular weight heparin or heparin contamination from a line draw
• 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
  • Antiphospholipid antibodies/lupus anticoagulant
  • Malignancy
  • Long-distance travel
  • Trauma
  • Surgery
  • Immobilization
  • Presence of a central venous catheter
  • Pregnancy/postpartum
  • Therapy-related thrombophilia
    • Hormone replacement therapy
    • Oral contraceptives
    • Tamoxifen/raloxifene
    • Chemotherapy
    • Heparin-induced thrombocytopenia

Screening

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 (heterozygous with factor V Leiden)	0.06-0.12	10.0	10
      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 factor V 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

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 and with no anticoagulant activity
• Inherited protein S deficiency is uncommon
  • Two forms
    • Type I – quantitative
    • Type II – qualitative
    • Type III – 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
  • C677T and A1298C most common
    • C677T 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

Click the plus sign to expand the table of additional tests.

Test Name and Number	Comments
APC Resistance Profile 0030127 Method: Clotting	Initial screen for suspected thrombophilia 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 not due to a factor V mutation will not be detected
Prothrombin (F2) G20210A Mutation 0056060 Method: Polymerase Chain Reaction/Fluorescence Monitoring	Detect prothrombin G20210A mutation
Antithrombin, Enzymatic (Activity) 0030010 Method: Chromogenic Assay	Detects both quantitative and qualitative deficiency of antithrombin
Antithrombin Panel 0030370 Method: Chromogenic Assay/Microlatex Particle-Mediated Immunoassay	Detect and type AT deficiency
Antithrombin, Antigen 0030015 Method: Microlatex Particle-Mediated Immunoassay	Not recommended to detect AT deficiency; use if necessary to distinguish quantitative from qualitative AT deficiency Functional test should be ordered first
Protein C and S Panel, Functional 0030182 Method: Clotting	Acceptable panel to detect protein C and S deficiencies
Protein C, Functional with Reflex to Protein C, Total Antigen 0030041 Method: Clotting/Enzyme-Linked Immunosorbent Assay	Detects both quantitative and qualitative deficiency of protein C If protein C is decreased, protein C, total antigen, will be added
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	Detects quantitative protein S deficiency and most forms of qualitative protein S deficiency
Protein S, Functional 0030114 Method: Clotting	Protein S free is the recommended initial test Acceptable test for detecting protein S deficiency Patient should be off of oral anticoagulants for two weeks for accurate measurement of functional protein S
Protein S, Total Antigen 0030112 Method: Microlatex Particle-Mediated Immunoassay	Not recommended for detecting protein S deficiency; protein S free is the recommended initial test Aid in the diagnosis of suspected protein S deficiency Patient should be off of oral anticoagulants for two weeks for accurate measurement of protein S
Protein C, Functional 0030113 Method: Clotting	Detects both quantitative and qualitative deficiency of protein C
Protein C, Total Antigen 0030111 Method: Enzyme Immunoassay	Not recommended for detecting protein C deficiency; functional  test should be ordered first Use if necessary to distinguish quantitative from qualitative protein C deficiency Patient should be off of oral anticoagulants for two weeks for accurate measurement of protein C
Homocysteine, Total 0099869 Method: Quantitative Enzymatic	Acceptable screen for disorders of methionine metabolism
Methylenetetrahydrofolate Reductase (MTHFR) 2 Mutations 0055655 Method: Polymerase Chain Reaction/Fluorescence Monitoring	Total homocysteine is the recommended first-line test Not recommended for nonsymptomatic patients <18 years of age
Factor VIII, Activity 0030095 Method: Clotting	Order to diagnose hemophilia A or as part of diagnostic workup for vWD
Factor V, R2 Mutation 2001549 Method: Polymerase Chain Reaction/Restriction Enzyme Digestion/Gel Electrophoresis	Further clarify thrombotic risk for individuals who are known FVL heterozygotes Mutations other than R2 (A4070G) are not evaluated by this assay Factor V, R2 mutation by itself does not significantly contribute to venous thrombosis risk
Factor XIII (F13A1) V34L Variant 2003220 Method: Polymerase Chain Reaction/Fluorescence Monitoring	Detect presence or absence of V34L variant Presence of variant associated with decreased/reduced risk for deep vein thrombosis, myocardial infarction and coronary artery disease Mutations other than the V34L sequence variant are not evaluated
Thrombotic Risk, DNA Panel 0056200 Method: Polymerase Chain Reaction	Acceptable panel to detect two most common inherited thrombophilias
Factor V Leiden (F5) R506Q Mutation 0097720 Method: Polymerase Chain Reaction/Fluorescence Monitoring	Detect F5 mutation