Hypercoagulable States - Thrombophilia

Diagnostic Algorithm

Clinical Background

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

Epidemiology

• Prevalence
  • Inherited thrombophilic defects found in 30-50% of venous thromboembolic events in U.S.

    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 FVL)	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	0.16-0.21	2.3	5.0
    Antithrombin deficiency	0.02	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 Adapted from Perry, Ortel, and references included within
    Whitlatch, NL and Ortel TL.  Thrombophilias: When should we test and how does it help?  Seminars in respiratory and critical care medicine. 2008; 29(1):27.

Etiologies

• Most-common thrombophilias
  • Factor V Leiden  
  • Prothrombin G20210A
  • Homocysteinemia (acquired or inherited)
• 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
  • Antithrombin deficiency
  • Impaired clot lysis (dysfibrinogenemia, abnormal fibrinolysis)
• Antiphospholipid syndrome is an acquired thrombophilic state – refer to the Antiphospholipid Syndrome topic in ARUP Consult

Select topics are discussed below

Factor V Leiden

• Genetics and pathophysiology
  • The factor V Leiden (FVL) point mutation 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 as a screening test for FVL
      • DNA tests are used to confirm positive screening tests and to differentiate between heterozygotes and homozygotes
  • Autosomal dominant inheritance
    • Heterozygous carriers have a 5- to 10-fold increased risk
    • Homozygous carriers have a 50- to 100-fold increased risk
• Clinical Presentation
  • Venous thromboembolism (VTE) is the most common type of thrombotic event
    • Recurrent VTE is generally uncommon in heterozygous patients unless additional risk factors are present
    • Risk of recurrent VTE is increased in homozygous carriers
  • Recurrent miscarriage in the second trimester of pregnancy
• Additional risk factors
  • Presence of factor V R2 A4070G mutation in addition to FVL mutation increases risk of thrombotic event 10-fold
  • Many patients with FVL mutation and recurrent episodes of thrombosis have more than one genetic risk factor (eg, concomitant prothrombin [factor II] G20210A mutation, protein C deficiency, homocystinemia)
  • Acquired factors such as pregnancy, oral contraceptives, hormone replacement therapy, and immobilization increase the risk

Prothrombin G20210A

• Genetics and pathophysiology
  • The prothrombin G20210A mutation is the second most common inherited thrombophilia
    • Results in elevated levels of plasma prothrombin which leads to hypercoagulability (gain of function)
    • Detected using DNA tests
      • Factor II (prothrombin) activity is not an appropriate test
  • Autosomal dominant inheritance
    • A single copy of the G20210A mutation increases the lifetime risk of venous thrombosis by 3-11% while possessing two copies of the mutation leads to even greater risk
• Clinical Presentation
  • VTE
  • Pregnancy complications
• Additional risk factors
  • Combined heterozygosity for the prothrombin G20210A mutation and FVL leads to earlier onset, higher rate of recurrence and more severe thrombotic events than either by itself
  • Risk of thrombosis appears increased during pregnancy and with oral contraceptive use

Protein C Deficiency

• Pathophysiology
  • Protein C is a vitamin K-dependent plasma anticoagulant that inactivates factors Va and VIIIa after being activated to APC by thrombin-thrombomodulin
    • Inherited protein C deficiency is uncommon and may be either quantitative (type I) or qualitative (type II)
      • Autosomal dominant inheritance 
      • Functional assays (rather than antigenic assays) are preferred for diagnosis
      • 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
      • Elevated FVIII levels (acute phase reactant) may interfere in some functional assays and result in falsely decreased values
    • Increased protein C levels may be seen in diabetes, nephrotic syndrome, during pregnancy, and in patients on oral contraceptives 
      • Heparin and direct thrombin inhibitors may interfere in some functional assays, resulting in falsely elevated values
• Clinical Presentation
  • Additional risk factors likely necessary to provoke thrombosis
  • VTE in heterozygotes
  • Neonatal purpura fulminans (DIC) in homozygous infants
  • Warfarin-induced skin necrosis is seen rarely

Protein S Deficiency

• Pathophysiology
  • Protein S is a vitamin K-dependent plasma anticoagulant which acts as a cofactor for activated protein C
  • Protein S exists in 2 forms
    • Free protein S represents 40% of the total and is physiologically active
    • Bound protein S (attached to C4b-binding protein) represents 60% of the total and possesses no anticoagulant activity
  • Inherited protein S deficiency is uncommon and may be either quantitative (type 1) or qualitative (type 2)
    • Autosomal dominant inheritance
    • Antigenic tests for free protein S are 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), disseminated intravascular coagulation (DIC), liver disease, malnutrition (vitamin K deficiency), pregnancy, estrogen therapy, and with warfarin therapy
    • Elevated FVIII levels may interfere in some functional assays and result in falsely decreased values
    • APC resistance may interfere in some functional assays and result in falsely decreased values
    • Heparin and direct thrombin inhibitors may interfere in some functional assays and result in falsely elevated values
• 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 is seen rarely

Antithrombin Deficiency

• Pathophysiology
  • Antithrombin (AT) is a plasma anticoagulant that inactivates thrombin, factor Xa and other activated clotting factors
    • Antithrombin activity is enhanced by heparin-like glycosaminoglycans on the endothelial surface and by pharmaceutical heparin
  • Inherited antithrombin deficiency may be either quantitative (type 1) or qualitative (type 2)
    • Autosomal dominant inheritance
    • Functional assays are preferred for diagnosis
  • Decreased antithrombin 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 in some patients
• Clinical Presentation
  • VTE
  • Recurrent thrombosis may occur even in the absence of additional risk factors
  • Some deficient patients are resistant to heparin therapy

Hyperhomocysteinemia

• Independent risk factor for thromboembolic events
• Most patients with hyperhomocysteinemia do not have genetic mutations or polymorphisms
• Regardless of the underlying etiology, hyperhomocysteinemia is the result of deranged homocysteine metabolism which may be acquired (deficiency of vitamins B6, B12, or folic acid) or inherited (deficiency of cystathionine β-synthase or expression of a thermolabile form of methylenetetrahydrofolate reductase)
• Thrombotic risk is most closely associated with increased fasting plasma homocysteine levels regardless of the underlying etiology
  • Plasma homocysteine testing is recommended rather than DNA-based tests

Methylenetetrahydrofolate reductase (MTHFR) mutations

• Genetics
  • Autosomal recessive inheritance
  • The most common genetic defects of homocysteine metabolism are the MTHFR mutations C677T and A1298C
    • The C677T mutation results in a thermolabile variant of MTHFR
• Clinical Presentation
  • Elevated plasma homocysteine levels have been associated with atherosclerotic disease, VTE and arterial thrombosis

Click here for diagram of Clotting Cascade with an Emphasis on Inherited Thrombophilias

Diagnosis

• Laboratory testing
  • Consider acquired disorders such as
    • APS – refer to Antiphospholipid Syndrome topic in ARUP Consult
    • Homocysteine – refer to Cardiovascular Disease, Non-traditional Risk Markers topics in ARUP Consult
    • Cancer screening based on age and risk factors
  • If factor V Leiden mutation found, consider testing the following (based on recommendation of the American College of Medical Genetics)
    • Prothrombin gene mutation G20210A
    • Factor V gene R2 mutation
    • Measurement of total plasma homocysteine concentration
    • Functional coagulation assays for antithrombin III, protein C and protein S deficiencies
• Indications for testing for inherited disorders
  • Population screening is not recommended
  • Patient with new VTE or recurrent VTE without obvious acquired risk factors (see Differential Diagnosis)
    • 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
    • Idiopathic thrombosis in patient ≤50 years of age
    • Recurrent thrombosis
    • Unusual sites of thrombosis
    • First-degree relatives with thromboses
    • Thrombotic event during pregnancy
    • Thrombotic event while taking oral contraceptives
  • If testing indicated, consider the following 
    • Activated protein C resistance (with or without reflex to FVL mutation); Factor V R2 A4070G mutation
    • Prothrombin mutation
    • Antithrombin activity
    • Protein C activity
    • Free protein S
    • Factor VIII activity (testing other factor activities such as FVIII and FIX is controversial and not currently recommended)
    • Testing for less common disorders is available if results are uninformative and additional testing is indicated
  • Many tests are altered by acute thrombotic states (acute phase response, consumption of factors and anticoagulant factors) and anticoagulant therapie
    • Delay testing 2-3 months after acute event
      • Preferable to 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
    • DNA-based tests are not affected by an acute phase response or anticoagulant therapy
  • Consider repeating abnormal functional or antigenic testing before making a definitive diagnosis of an inherited thrombophilia
    • Low results can be obtained due to patient condition//biologic variability, medications, and assay variability or interference
    • Consider patient age and gender when interpreting results; normal ranges vary by age and gender

Differential Diagnosis

• Provoked/acquired causes of thrombophilia are more common than hereditary causes and should be considered when evaluating patients with thrombosis.
• Examples of provoked/acquired causes of thrombophilia include:
  • 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

Pharmacogenetics and Therapeutic Drug Monitoring

Screening

Monitoring

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	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
Antithrombin, Antigen 0030015 Method: Microlatex Particle-Mediated Immunoassay	Functional test should be ordered first Use if necessary to distinguish quantitative from qualitative AT deficiency
Protein S, Functional 0030114 Method: Clotting	Protein S free is the recommended initial test Aid in the diagnosis of suspected protein S deficiency
Protein S, Total Antigen 0030112 Method: Microlatex Particle-Mediated Immunoassay	Protein S free is the recommended initial test Aid in the diagnosis of suspected protein S deficiency
Protein C, Total Antigen 0030111 Method: Enzyme Immunoassay	Functional  test should be ordered first Use if necessary to distinguish quantitative from qualitative protein C deficiency
Methylenetetrahydrofolate Reductase Mutation Detection (Thermolabile Form) (C677T & A1298C) 0055655 Method: Polymerase Chain Reaction/Fluorescence Monitoring	Not recommended for nonsymptomatic patients <18 years of age. Total homocysteine is the recommended first-line test Analytical sensitivity and specificity – 99%
Thrombotic Risk, DNA Panel 0056200 Method: Polymerase Chain Reaction
Antithrombin Panel 0030370 Method:
Protein C & S Panel, Functional 0030182 Method: Clotting
Protein C & S Panel, Total, Antigen 0030116 Method: Microlatex Particle-Mediated Immunoassay

Guidelines

General References

References from the ARUP Institute for Clinical and Experimental Pathology®