Thrombophilia laboratory findings

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Editor-In-Chief: C. Michael Gibson, M.S., M.D. [1] Associate Editor(s)-in-Chief: Asiri Ediriwickrema, M.D., M.H.S. [2] Jaspinder Kaur, MBBS[3]

Overview

Laboratory findings consistent with the diagnosis of inherited thrombophilias vary based on the etiology of the thrombus.

Laboratory Findings

Initial assessment

• The initial assessment of a patient presenting with a clinical picture suggestive of thrombophilia must commence with a carefully taken personal and family history. In addition, a clinical examination with appropriate laboratory, imaging and other investigation should be performed. Depending on the initial assessment of the patient and on the clinical management decisions to be addressed, laboratory testing for heritable thrombophilia can be considered.
• The most recent NICE guidelines recommend against offering inherited thrombophilia testing to patients presenting with a provoked VTE in any clinical setting. In patients diagnosed with unprovoked VTE, testing should not be considered unless a first degree relative with a history of VTE exists. The NICE guidelines also recommend against routinely offering thrombophilia testing to asymptomatic first-degree relatives of patients with a history of VTE or known inherited thrombophilia. This recommendation is reflected in the American Society of Hematology’s Choosing Wisely recommendations since 2013. The available evidence suggests that clinicians should avoid ordering thrombophilia testing for hospitalized patients with unprovoked VTE because (1) many thrombophilia tests are inaccurate in the setting of acute VTE and/or anticoagulation, (2) results of testing often do not influence management, (3) testing is not cost-effective, (4) a positive test result may lead to unnecessary patient anxiety, and (5) testing may result in inappropriately prolonged anticoagulation courses or unnecessary involvement of inpatient consultants.
• The available evidence suggests that testing for inherited thrombophilia is not recommended in most clinical settings. In patients without a personal history of VTE, thrombophilia results do not change management, as there is no evidence to support thromboprophylaxis in this setting. In patients with a personal history of provoked or unprovoked VTE, inpatient testing is not indicated, as results do not influence management, testing is not cost-effective, and a positive test result may lead to unnecessary patient anxiety or may result in unnecessary involvement of consultants. Testing in hospitalized patients has even more limitations because many thrombophilia tests are inaccurate in the setting of acute VTE and/or anticoagulation. Depending on the underlying condition and expression of the genetic abnormality, the relative risk of VTE in patients with inherited thrombophilia is 3- to 20-fold greater than that of the general population. Therefore, it is logical to consider that testing for inherited thrombophilia might be clinically useful. thrombophilia testing may divert attention away from the management of more prevalent, potentially modifiable risk factors such as immobility, oral contraceptive use, or malignancy, all of which are associated with recurrent VTE. However, the evidence for doing so is very limited. Testing should only be considered using an individualized approach in the outpatient setting with appropriate genetic counseling.
• Determining whether a blood clot classifies as provoked (most common) vs. unprovoked, and whether it is the first episode vs. subsequent are critical aspects of the initial evaluation that can guide further workup and treatment. Clinicians should take a careful personal and family history to document any thrombosis history or pregnancy morbidity. In carefully selected patients suspected of having an underlying hereditary thrombophilia, a targeted hypercoagulable workup may be indicated. Such testing may include checking the amount of protein C and S, antithrombin III, or mutational testing for factor V Leiden or Prothrombin gene mutation. Acquired thrombophilia testing may also be indicated, such as antiphospholipid antibody testing. In general, a full hypercoagulable workup is rarely necessary and strongly discouraged on initial presentation in the absence of subspecialty evaluation with a hematologist; this is especially true in the acute setting, as many factors can affect the accuracy of test results (e.g., acute inflammation or consumption of coagulation factors due to acute thrombosis can lead to falsely low levels of some coagulation factors). Anticoagulation therapy can also interfere with the interpretation of test results in the acute setting (e.g., heparin can affect the interpretation of Antithrombin III). For antiphospholipid antibody testing, many medications (including several anticoagulants), as well as underlying connective tissue diseases such as SLE, can affect the testing result and interpretation of the lupus anticoagulant. When antiphospholipid antibody testing is indicated, the recommendation is that the tests be repeated 12 weeks apart. As such, clinicians should reserve such hypercoagulable testing to carefully selected patients (e.g., young patients with unprovoked thrombosis or in those with confirmed or strong family history thrombophilia). This testing is often performed in the outpatient setting after the acute phase of the thrombosis has resolved and under the guidance of a hematologist consultant.

Indications for thrombohilia screening and testing

• The most recent NICE guidelines recommend against offering inherited thrombophilia testing to patients presenting with a provoked VTE in any clinical setting. In patients diagnosed with unprovoked VTE, testing should not be considered unless a first degree relative with a history of VTE exists. The NICE guidelines also recommend against routinely offering thrombophilia testing to asymptomatic first-degree relatives of patients with a history of VTE or known inherited thrombophilia. This recommendation is reflected in the American Society of Hematology’s Choosing Wisely recommendations since 2013. The available evidence suggests that clinicians should avoid ordering thrombophilia testing for hospitalized patients with unprovoked VTE because (1) many thrombophilia tests are inaccurate in the setting of acute VTE and/or anticoagulation, (2) results of testing often do not influence management, (3) testing is not cost-effective, (4) a positive test result may lead to unnecessary patient anxiety, and (5) testing may result in inappropriately prolonged anticoagulation courses or unnecessary involvement of inpatient consultants.

• Indications: Thrombophilia testing may be considered in the following scenarios:
  • A person with venous blood clots in the legs or lungs (DVT or PE) in the “unclear how long” group in Figure 1:
    • Clot associated with only a mild trigger (minor surgery, minor immobility or short-distance travel, birth-control pill, patch or ring).
    • Clot that is unprovoked (idiopathic) but where the person is (i) at increased risk for bleeding or (ii) has a strong preference not to be on a blood thinner.
  • A person with an unexplained blood clot in a vein in an unusual location (such as in the veins of the abdomen or surrounding the brain).
  • A person who has never had a clot, but who has a first-degree relative with a strong thrombophilia (mother, father, sister, brother, child).
  • Unexplained arterial blood clot in a young person.
  • Recurrent miscarriages with no other cause.
  • Patient requests testing: Patient with a blood clot in the legs or lungs that is unprovoked or due to a minor risk factor who requests testing to understand why he/she developed a clot.

• Indications for Venous Thrombophilia Screening:
  • Age <50 years
  • Women with VTE during pregnancy or puerperium
  • Women with VTE during use of oral contraceptive or hormonal replacement
  • Women with VTE before prescribing hormonal replacement
  • Women with multiple inexplicable pregnancy losses
  • Young women with a positive family history, before prescribing oral contraceptive
  • VTE in unusual sites
  • First VTE and a positive family history for VTE
  • Young patients with arterial ischemia and right-to-left shunt (paradoxical embolism)

• WHO SHOULD BE TESTED?: Recommendations on selection of patients for thrombophilia case finding: Indiscriminate application of laboratory investigations is clinically inappropriate, a waste of scarce resources and can be misleading. Diagnostic uncertainty is frequent.
  • Testing of unselected patients is inappropriate and should be avoided.
  • Haematologists must give clear guidance to clinical colleagues on the selection of patients for testing and should oversee requests.
  • There is little if any clinical value in testing for heritable thrombophilia if the appropriate mechanisms for tracing, careful informed counselling and testing of at risk relatives are not in place, as there is a risk of engendering confusion, misinformation, false reassurance and unnecessary anxiety.
  • In relation to heritable thrombophilia, in most instances any value of laboratory testing will relate to the possibility of preventing a first venous thromboembolic event in affected relatives. The effectiveness and risks of this approach have not been formally assessed. The potential for any such benefit should form part of the criteria for testing. For example, testing for thrombophilia is unlikely to be informative in an elderly subject with a first venous thromboembolism in whom the family history is negative. In contrast, where unprovoked venous thrombosis occurs in a subject with a positive family history or in a young subject with children or siblings, especially female children or women of child-bearing age, identification of heritable thrombophilia may assist in counselling of affected relatives regarding the avoidance of risk. In adopting this approach it must be borne in mind that, in most instances, heritable thrombophilia represents a non-life-threatening late-onset genetic disorder that will not manifest clinically in a large proportion of affected individuals. The presence of a strongly positive family history of thrombosis may serve as a useful indicator of possible benefit from counselling and case-finding.
  • Testing for heritable defects and, in particular, genetic testing should be avoided in children unless there is a very strong clinical indication for it.
  • Although it is widely accepted that women with a history of three or more consecutive pregnancy losses should be screened for antiphospholipid antibodies, it would be premature to recommend that testing of these women or women with a history of pre-eclampsia or intrauterine growth retardation should be extended to include testing for heritable defects.

Common thrombophilia tests

• Testing for V Leiden: Activated protein C (APC) resistance test and/or factor V Leiden genetic test
  • Prothrombin (factor II) G20210A genetic test
  • Protein C activity
  • Protein S activity, free protein S antigen
  • Antithrombin activity
  • Antiphospholipid antibodies: Anticardiolipin antibodies, Anti-beta-2-glycoprotein I antibodies, Lupus anticoagulant
  • Other tests: In patients with unexplained clots in the abdomen: JAK-2 mutation, PNH test
  • In young patients (<30 years old) with unexplained vein or artery clots: homocysteine
  • Not to obtain: MTHFR genetic test; factor VIII level; tPA and PAI-1 blood levels or genetic tests
  • Timing of testing for thrombophilia is important because results can be falsely abnormal at the time of the initial clotting episode or while on a blood-thinner.
  • JAK, Janus kinase 2; PNH, paroxysmal nocturnal hemoglobinuria; MTHFR, methylenetetrahydrofolate reductase; tPA, tissue plasminogen activator; PAI-1, plasminogen activator inhibitor type 1.
  • Acquired thrombophilias and preferred approach to diagnostic workup as follows:
  • Surgery, trauma, immobility, hospitalization, indwelling catheter, high estrogen state: History (transient/reversible risk factors)
  • Myeloproliferative neoplasm: Mutation analysis for JAK2, CALR, MPL
  • Malignancy, SLE/collagen vascular disease, nephrotic syndrome, inflammatory bowel disease, obesity: History/examination, basic laboratory tests (CBC, renal/ hepatic panels, urinalysis for protein), chest radiograph, age-appropriate cancer screening
  • Paroxysmal nocturnal hemoglobinuria: If suspected, CBC, haptoglobin, LDH, total/direct bilirubin, iron studies, urinalysis; peripheral blood flow cytometry
  • APLS: Revised Sapporo criteria (both required): clinical, vascular thrombosis and/or pregnancy morbidity; laboratory, 1 of the following on $2 occasions at least 12 weeks apart: IgG or IgM anti-cardiolipin antibodies (.40 U); IgG or IgM anti-b2-glycoprotein I antibodies (.40 U); LA

Recommendations for laboratory tests and interpretation

• When testing is indicated, it should include assays for heritable defects: deficiency of antithrombin, protein C or protein S, factor V Leiden and prothrombin G20210A mutations and for antiphospholipid antibodies.
  • The activated partial thromboplastin time (APTT), prothrombin time and thrombin clotting time should be incorporated in the initial screening. The APTT may identify some patients with antiphospholipid antibodies (depending on the sensitivity of the APTT reagent used), but is not sufficient alone to exclude antiphospholipid antibodies. The thrombin clotting time will allow identification of dysfibrinogenaemia and heparin contamination. The prothrombin time is useful in the interpretation of low protein C or protein S results.
  • Functional assays should be used to determine antithrombin and protein C levels
  • Chromogenic assays of protein C activity are less subject to interference than clotting assays and are preferable.
  • Immunoreactive assays of protein S antigen are preferable to functional assays. If a protein S activity assay is used in the initial screen, low results should be further investigated with an immunoreactive assay of free protein S.
  • The modified APC:SR test (predilution of the test sample in factor V-deficient plasma), as opposed to the original APC:SR test, should be used as a phenotypic test for the factor V Leiden mutation.
  • PCR-based testing for prothrombin G20210A is required, as there is no screening test
  • Laboratories must establish their own reference ranges for the assays and tests that they use, including antithrombin, protein C and protein S and modified APC:SR
  • Comprehensive assays for antiphospholipid antibodies (both lupus inhibitors and anticardiolipin antibodies) should also be performed.
  • Rigorous internal quality assurance and participation in accredited external quality assessment schemes are mandatory.
  • The interpretation of thrombophilia test results is difficult and fraught with pitfalls, which occasionally lead to underdiagnosis and frequently to overdiagnosis of defects.

It is strongly recommended that thrombophilia testing is supervised by and results are interpreted by an experienced clinician who is aware of all relevant factors that may influence individual test results in each individual.

Factors affecting the accuracy of the tests

• Warfarin decreases protein C and protein S levels.
  • Heparin decreases antithrombin activity and lead to false positive results for lupus anticoagulant.
  • Direct acting oral anticoagulants can interfere with evaluation for lupus anticoagulant, antithrombin activity, protein C, and S levels.
  • Acute thrombosis decreases antithrombin, protein C, and protein S levels.
  • Pregnancy, cirrhosis, nephrotic syndrome, chemotherapies (l-asparaginase), anticoagulants, disseminated intravascular coagulation, and systemic illness can alter coagulation factor levels.

When to collect samples for thrombophilia testing

• Some tests for heritable thrombophilia (for example, assays of antithrombin, protein C and protein S) are affected by the acute post-thrombotic state and by anticoagulant use. Also, finding a thrombophilic abnormality almost never influences the management of an acute thrombotic event. There is little point in striving to obtain samples for tests for heritable thrombophilia when the patient presents with an acute thrombotic event. Testing is usually best delayed until at least 1 month after completion of a course of anticoagulation. If possible, testing for heritable thrombophilia should be avoided during intercurrent illness, pregnancy, use of a combined oral contraceptive pill or hormone replacement therapy. If this is impossible, then it is essential that the individual interpreting the screen is aware of the presence and potential influence of these various acquired factors on the components of the test results. PCR-based tests for FV Leiden and the prothrombin 20210A allele are unaffected by the above factors.
  • Given the variability discussed above, timing is important to accurately evaluate for certain thrombophilic states.
  • Acute thrombosis can affect levels of coagulation factors, therefore, testing should be delayed for approximately six months.
  • Anticoagulation can affect certain tests, and should be completed approximately four weeks following completion of anticoagulation.
  • Avoid testing during severe illness.
  • Pregnancy, oral contraceptives, hormone replacement therapy, and cancer chemotherapy may also affect some tests.
  • Factor V Leiden and Prothrombin mutation can be done in patients on anticoagulants and even in acute phase, as these are genetic tests.

Emergency room assessment and screening

• When patients present to the emergency department with signs and symptoms suggestive of possible venous thrombosis (see previous section H&P findings), a well-validated scale known as the modified Wells' criteria is applied to help guide further diagnostic studies. For patients with high Wells score, a serum D-dimer should be checked. The D-dimer is a fibrin degradation product that is present in the blood after fibrinolysis. Its elevation is very sensitive (though less specific) to detect venous thrombosis. It is important to note that a D-dimer could also be elevated in other patients such as pregnant and post-surgical patients, or those with underlying malignancy. However, it aids clinicians in deciding whether to pursue further diagnostic imaging. A negative D-dimer result helps to rule out a clot and avoid unnecessary imaging studies or anticoagulation initiation. The pulmonary embolism rule-out criteria (PERC) is also occasionally applied to help decide whether the patient has developed an acute pulmonary embolism (PE), though not applicable for DVT. The modified Wells score can also be used if physicians suspect an acute DVT. When there is a high pretest probability for PE or DVT, imaging studies should be completed immediately without regard to D-dimer levels. For pulmonary embolism, the recommended imaging studies are CT angiography and ventilation/perfusion imaging (V/Q scan). The V/Q scan is sometimes preferred over CTPA to avoid radiation exposure or intravenous contrast in those with underlying renal impairment. However, not all facilities have V/Q scanning capabilities or expertise at interpreting the results, so CTPA is often used. Often, pulmonary emboli result from fragmentation of preexisting thrombosis in an extremity (i.e., DVT). Hence, compression sonography (Duplex US) of lower and/or upper extremities is also often performed to evaluate for concurrent DVT. This is especially important if a provoking catheter-related thrombosis is suspected, as the catheter may require eventual removal.

Protein C deficiency

• Diagnostic testing for protein C deficiency is performed using functional assays including clotting assays, enzyme-linked immunosorbent assays (ELISA) and chromogenic tests to determine levels of protein C activity. Mutational analysis of the PROC gene is also available.

Malignancy

• Malignancy can be an underlying provoking factor for thrombosis (both venous and arterial). In general, it is not recommended for patients to have an extensive malignancy workup in the absence of any clinical factors to suggest underlying malignancy as the provoking cause. Age-appropriate cancer screening is recommended for all patients. In carefully selected patients, especially in those over the age of 50 with seemingly unprovoked thrombosis, in whom malignancy is a possibility, further evaluation to search for occult malignancy as the culprit may be indicated.
• Occult cancer should be considered, although invasive investigation is not routinely indicated. A full blood count should be performed to exclude myeloproliferative disorders.