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

• Aim: 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.
• The initial assessment involves a carefully taken clinical history and physical examination in addition to performing appropriate laboratory, imaging and other relevant investigations.
• Depending on the initial assessment and the clinical management decisions to be addressed, laboratory testing for heritable thrombophilia can be considered.
• NICE guidelines:
  • Provoked VTE: The most recent guidelines recommend against offering inherited thrombophilia testing to patients presenting with a provoked VTE in any clinical setting.
  • Unprovoked VTE: Testing should not be considered unless a first degree relative with a history of VTE exists.
  • The NICE guidelines in accordance with the American Society of Hematology’s Choosing Wisely recommendations also recommend against routinely offering thrombophilia testing to asymptomatic first-degree relatives of patients with a history of VTE or known inherited thrombophilia as there is no evidence to support thromboprophylaxis in this setting.
• Hospitalized patients:
  • Its been recommended that the clinicians should avoid ordering thrombophilia testing for hospitalized patients with unprovoked VTE because of the following:
    • Many thrombophilia tests are inaccurate in the setting of acute VTE and/or anticoagulation
    • Results of testing often do not influence management
    • Testing is not cost-effective
    • A positive test result may lead to unnecessary patient anxiety
    • Testing may result in inappropriately prolonged anticoagulation courses or unnecessary involvement of inpatient consultants.

• Individualized approach: Depending on the underlying inherited 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 which further states that testing for inherited thrombophilia might be clinically useful. However, the testing process may divert attention away from the management of more prevalent and potentially modifiable risk factors such as immobility, oral contraceptive use, or malignancy which are also associated with recurrent VTE. However, the evidence for doing so is very limited. Hence, testing should only be considered using an individualized approach in the outpatient setting with appropriate genetic counseling.

Indications for thrombohilia screening and testing

• Indiscriminate application of laboratory investigations is clinically inappropriate, wastage of scarce resources, creates unnecessary anxiety, apprehension regarding recurrence, and can be misleading as diagnostic uncertainty is frequent.
• Hence, its been advisable to take hematologists consult and/or a genetic counselor to determine the risks and benefits of testing.

Table 1: Thrombophilia testing may be indicated in the following scenarios:

Indications for Thrombophilia testing

Age <50 years A person with venous blood clots in the legs (DVT) or lungs (PE) with the following conditions: Clot associated with a mild trigger such as minor surgery, minor immobility or short-distance travel, birth-control pill, patch or ring Clot that is unprovoked or idiopathic but the affected patient is at increased risk for bleeding or has a strong preference not to be on a blood thinner Patient requests testing to understand why he/she developed a clot A person with an unexplained blood clot in an unusual venous sites such as in the veins of the abdomen or surrounding the brain A person with no history of a clot but 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 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 First VTE and a positive family history for VTE Young patients with arterial ischemia and right-to-left shunt (paradoxical embolism)

Common thrombophilia tests

• An interpretation of thrombophilia test results is difficult and fraught with pitfalls which can occasionally lead to underdiagnosis and frequently to overdiagnosis of defects. Hence, 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.
• Additionally, the tests will not find abnormalities in all patients with VTE and a strong family history which reflects the likelihood that some heritable states are yet to be identified especially in the non-Caucasian population. Therefore, a negative set of investigations does not exclude an inherited prothrombotic tendency and a referral to a thrombosis specialist should be made in the doubtful cases.

• Table 2: Diagnostic workup for thrombophilia testing

Diagnostic tests

Initial screening: activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin clotting time APTT: Identify some antiphospholipid antibodies PT: Interpret low protein C or protein S levels Thrombin clotting time: Detect dysfibrinogenaemia and heparin contamination Factor V Leiden mutation: Activated protein C (APC) resistance test: A 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 Factor V Leiden genetic test Prothrombin (factor II) G20210A PCR-based genetic test Protein C deficiency: Functional assays such as clotting assays, enzyme-linked immunosorbent assays (ELISA) and chromogenic tests (preferable) to determine protein C activity levels Mutational analysis of the PROC gene Protein S activity level and free protein S antigen functional and immunoreactive assays Antithrombin activity level measured through functional assays Antiphospholipid antibodies: Anticardiolipin antibodies Anti-beta-2-glycoprotein I antibodies Lupus anticoagulant Young patients (<30 years old) with unexplained clots: Homocysteine levels Surgery, trauma, immobility, hospitalization, indwelling catheter, high estrogen state: History (transient/reversible risk factors) Myeloproliferative neoplasm: Mutation analysis for JAK2 (Janus kinase 2), 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, and/or age-appropriate cancer screening Cancer screening: Routine (history and physical examination, ESR, CBC, liver and kidney function tests, urinalysis, and CXR) or extended investigations (tumor markers, CT of the chest, abdomen, and pelvis, mammography in women more than 40 years old, and prostate ultrasound in men older than 50 years, lower endoscopy, Papanicolaou smear, and fecal occult blood test). Paroxysmal nocturnal hemoglobinuria: If suspected, CBC, haptoglobin, LDH, total/direct bilirubin, iron studies, urinalysis and peripheral blood flow cytometry Antiphospholipid antibodies: Revised Sapporo criteria (both required): clinical, vascular thrombosis and/or pregnancy morbidity; Laboratory with 1 of the following on more than 2 occasions at least 12 weeks apart: IgG or IgM anti-cardiolipin antibodies (.40 U) or IgG or IgM anti-b2-glycoprotein I antibodies (.40 U)

• Following tests are not indicated commonly:
  • MTHFR (methylenetetrahydrofolate reductase) genetic test
  • Factor VIII level
  • tPA (tissue plasminogen activator) blood levels
  • PAI-1 (plasminogen activator inhibitor type 1) blood levels
  • Genetic tests

Factors affecting the accuracy of the tests

• Table 3: List of factors affecting the accuracy of thrombophilia test results

Factors

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 protein S levels Acute inflammation or consumption of coagulation factors due to acute thrombosis can lead to falsely low levels of antithrombin, protein C, and protein S Pregnancy, cirrhosis, nephrotic syndrome, chemotherapies (l-asparaginase), anticoagulants, disseminated intravascular coagulation, connective tissue diseases such as SLE and systemic illness can alter coagulation factor levels Medications including anticoagulants, oral contraceptives, hormone replacement therapy, and cancer chemotherapy may affect the test outcomes. Tests conducted at the time of the initial clotting episode or while on a blood-thinner can produce falsely abnormal results

When to collect samples for thrombophilia testing

• As the management of an acute thrombotic event never gets influenced by the finding of a thrombophilic abnormality. Hence, there is little point in striving to obtain samples for tests for heritable thrombophilia when the patient presents with an acute thrombotic event.
• Timing of testing:
  • Testing should be done approximately four weeks following the completion of a course of anticoagulation.
    • Vitamin K antagonists: Testing sample should be taken 2 weeks after their cessation
    • Low molecular weight heparins: Tests should be performed 24 hours after stopping them as these produce a false positive lupus anticoagulant test.
  • Testing should be delayed for approximately six months in acute thrombosis events which can affect levels of coagulation factors or once the acute phase of the thrombosis has resolved.
  • Antiphospholipid antibody testing is recommended to be repeated 12 weeks apart.
• PCR-based tests for FV Leiden and the prothrombin 20210A allele mutation can be done in patients presenting in an acute phase and/or being on anticoagulants as these are genetic tests.
• Sample collection:
  • All samples should be taken in sodium citrate/blue vacutainers filling them to the line to ensure the correct dilution is attained.
  • Local policies for anticardiolipin tests vary and may require a clot-activated sample with red top vacutainer or without gel for serum separation/gold top vacutainer.
• Hence, the timing and sample collection are important to accurately evaluate for the thrombophilic states given the variability of test results under the guidance of a hematologist consultant.

Emergency room assessment and screening

• Modified Wells' criteria: It is applied to the patients presenting to the emergency department with signs and symptoms suggestive of possible venous thrombosis to help guide further diagnostic studies. For patients with high Wells score, a serum D-dimer should be checked.
• D-dimer: It is a fibrin degradation product that is present in the blood after fibrinolysis.
  • Positive: Its elevation is very sensitive though less specific to detect venous thrombosis; however, it could be elevated in patients with underlying malignancy, post-surgical patients and pregnant females.
  • Negative: A negative D-dimer result helps to rule out a clot and avoid unnecessary imaging studies or anticoagulation initiation.
• However, when there is a high pretest probability for PE or DVT, imaging studies should be completed immediately without regard to D-dimer levels.
• 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.
• Deep venous thrombosis: As often, pulmonary emboli result from fragmentation of preexisting thrombosis as a DVT in an extremity. Hence, compression sonography (Duplex US) of lower and/or upper extremities is also often performed to evaluate for concurrent DVT.