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]

Overview

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

• 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.

• Acquired thrombophilias and preferred approach to diagnostic workup:
  • 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

Laboratory Findings

Variability in thrombophilia testing

• 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.

Timing

• 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.

• 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.

Type of tests

Tests for thrombophilia are categorized according to their priority, as summarized below.

• High priority testing should be conducted if thrombophilia screening conditions are met.
• Intermediate and low priority testing should be completed in the appropriate clinical context.

Priority	Timing	Test	Associated Diagnosis
High		Complete blood count	General
		Coagulation studies: INR, prothrombin time, partial thromboplastin time	General
		Factor V Leiden mutation studies	Factor V Leiden
		Prothrombin 20210 gene mutation	Prothrombin G20210A
		Lupus anticoagulant	Antiphospholipid syndrome
		Anticardiolipin antibodies	Antiphospholipid syndrome
	Delay 6 months	Functional assay for antithrombin (antithrombin-heparin co-factor assay)	Antithrombin deficiency
	Delay 6 months	Protein C functional assay (preferred over plasma protein levels)	Protein C deficiency
	Delay 6 months	Free protein S immunoassay	Protein S deficiency
Intermediate		Factor VIII level (use c-reactive protein as control)	Factor VIII disorders
		Flow cytometry	Paroxysmal nocturnal hemoglobinuria
		Peripheral blood smear, JAK2 mutation studies	Myeloproliferative disorders
		Homocysteine level	Hyperhomocysteinemia
		Vitamin B12 level	Hyperhomocysteinemia
Low		Thrombin time	General/Fibrinogen disorders
		Fibrinogen level	Fibrinogen disorders
		Reptilase time	General/Fibrinogen disorders
		Plasminogen level	Plasminogen disorders
		Factor IX activity	Factor IX disorders
		Factor X activity	Factor X disorders

• 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.
• Emergency room: 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.
• 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.
• 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.

• 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.

• 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.

• 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)