Thrombophilia Testing in the Era of Direct Oral Anticoagulants: Clinical Recommendations

Thrombophilia is a condition in which the patient’s blood has an increased tendency to clot. Typically, the first presentation is venous thromboembolism (VTE). Thrombophilias can be inherited or acquired and confer different coagulation risks, depending on the type.

Despite the increased risk of thrombosis, there is no general consensus on when thrombophilia testing should be performed. In the last decade, the use of direct oral anticoagulants (DOACs) has seen an exponential increase; however, they have been shown to affect coagulation tests used to detect thrombophilias, especially those used to evaluate antiphospholipid syndrome (APS).

Types of thrombophilia

Hereditary thrombophilias can be classified as high or low risk.

High-risk thrombophilias are due to inherited deficiencies in endogenous anticoagulants, including protein C and protein S and antithrombin (AT) deficiencies, and also combined thrombophilic defects. Other examples include paroxysmal nocturnal hemoglobinuria (PNH) and patients with JAK2-positive myeloneoplasias.

Low-risk thrombophilias include mutations of factor V Leiden (FVL) and the prothrombin time (PT) gene G20210A. Other examples of low risk are factors VIII, IX and XI; elevated, plasminogen activator inhibitor; dysfibrinogenemia and hyperhomocysteinemia, but they are not routinely tested for thrombophilia scoring and are therefore not discussed in detail here.

APS is an acquired thrombophilia and confers a high risk of VTE as well as arterial thrombosis. It is characterized by the presence of lupus anticoagulant (LA) ß-2 glycoprotein and anticardiolipin antibodies. Patients with APS who have all 3 antibodies (triple positivity) have the highest risk of VTE.

Who requires thrombophilia testing?

There is general agreement that most patients suffering from VTE should not be tested for thrombophilia, but only in selected patients.

It has been proposed that inherited thrombophilias can be detected by examining a family and personal history of VTE, without the need for a laboratory test. Patients with hereditary thrombophilias often have key characteristics in their history.

It has been proposed that all patients with a personal history of VTE ≥50 years and a strong family history of VTE should be tested for thrombophilia. However, different specialized guides suggest the opposite.

Hereditary thrombophilias

In 2013, the American Society of Haematology’s ’Choosing Wisely’ campaign emphasized that patients presenting with VTE with significant transient risk factors (eg, recent surgery, malignancy, pregnancy) should not undergo thrombophilia testing.

However, most of the most recent guidelines for VTE management, published in 2018, do not comment on thrombophilia testing. Similarly, in its 2016 VTE update, the American College of Chest Physicians does not provide guidance on screening for thrombophilia, while the 2012 British Compete for Standards in Haematology document on inherited thrombophilia testing for patients and families , says that it is not possible to make a validated recommendation about which patients should be selected for testing. There is also no mention of how to perform a thrombophilia test when the patient is taking a DOAC.

The International Union of Angiology guideline for thrombophilia testing is more detailed and specific; however, it does not mention how to perform thrombophilia testing if the patient is taking a DOAC. Other guidelines suggest not testing for thrombophilia in those on continuous anticoagulant therapy or who have had a provoked VTE.

The NICE VTE (National Institute of Clinical Excellence) VTE guideline does not offer thrombophilia testing for those on continuous anticoagulant therapy or who have had an unprovoked VTE. If the patient has an unprovoked VTE and anticoagulants have been stopped, testing can be done. antiphospholipid antibody testing, but if the patient has unprovoked VTE and 1 first-degree relative with a history of VTE, testing for antiphospholipid antibodies can be done. Routine screening for first-degree relatives is not recommended.

NICE recommends that, for unprovoked VTE, the risk of clotting and bleeding should be established and decision-making shared with the patient. It does not recommend the use of risk scores alone to assess the risk of thrombosis, but suggests using tools such as the HAS-BLED score for the assessment of risk of major bleeding (although HAS-BLED has not been validated in a context other than atrial fibrillation.

Risk scores

With respect to the risk of recurrence of an unprovoked VTE, an assessment of coagulation vs. the risk of bleeding. There are scoring systems to assess clotting risk; but many of them– e.g. DASH, Vienna prediction model, and HERDOO2–do not take thrombophilia status into account, therefore the risk of recurrence cannot be accurately calculated in patients with thrombophilia.

There are also several risk scores to assess the risk of bleeding in patients who are anticoagulated. Although NICE recommends using the HASBLED score to assess patients after a VTE, this has only been validated for atrial fibrillation, and assessments found in the literature of its quality and how they compare to each other are conflicting. Such scoring systems can therefore assist in informative conversations with patients, but should not be used in isolation for decision making.

It is important to keep in mind that when it comes to risk analysis; Those patients who have had an unprovoked VTE, without an underlying risk factor, have a much higher risk of recurrence than those patients with a provoked VTE. NICE recommends that all patients with unprovoked VTE should receive long-term anticoagulants, following a discussion of the risk/benefit balance.

NICE’s recommendation is that in those patients who have had a VTE or a provoked proximal pulmonary thromboembolism, anticoagulation can be interrupted, once the provoking factor has been eliminated, as long as 3 months of treatment have been completed.

Acquired thrombophilias

There are also several guidelines that consider thrombophilia testing for acquired thrombophilias. In 2014, the Clinical Laboratory and Standards Institute guidance explicitly states that LA testing is not recommended for patients receiving DOAC treatment. In 2009 and 2014, the Committee of the International Society on Thrombosis and Hemostasis published 2 sets of guidelines on antiphospholipid antibody testing.

The 2009 guidelines suggest that testing for AL could be performed after transitioning from transient low-molecular-weight heparin (LMWH), when the patient is taking a vitamin K antagonist (VKA) such as warfarin, but not no specific reference is made to DOACs. Since the 2014 guidelines do not refer to DOACs or VKAs, it could be inferred that the same criteria are valid for patients taking DOACs.

How do DOACs DOACs affect thrombophilia testing?

DOACs have been shown to affect coagulation assays using thrombophilia testing, especially those used for APS testing. However, the results of these tests appear accurate in the presence of DOACs, depending on their levels, suggesting that patients anticoagulated with DOACs could also be tested for thrombophilia.

Diluted Russell’s viper venom time (TVVRD) is a laboratory test often used to evaluate the presence of AL, and is a key component of the activated protein C resistance (APCR) test, which is can be used to evaluate inherited thrombophilias such as FVL deficiency. It is significantly affected by DOACs depending on the dose. Therefore, the prolongation of TVVRD by the action of DOACs causes prolongation of coagulation in the RPCA test.

There is also a variable prolongation of the test in patients taking dabigatran and rivaroxaban and a significant prolongation if taking apixaban. However, this is at a concentration level 50% higher than the upper maximum levels.

There are also false low RPCA results caused by DOACs, which leads to a false image similar to FVL. It has been recommended that this test can be performed by direct DNA detection methods, and, in fact, these are usually carried out when RPCA is consistent with FVL deficiency.

Genetic testing for FVL is not affected in patients already receiving DOACs, and will help predict risk more accurately, depending on the mutation present and whether the patient is homozygous. Laboratory tests to detect thrombophilias include 1-step coagulation tests or 2-step chromogenic tests.

Chromogenic tests for protein C levels and immunoassay tests for protein S levels are not affected by the use of DOACs, but the same cannot be said for coagulation tests.

Factor _ _ of hereditary thrombophilia in patients taking DOACs.

To combat the effects of factor Xa inhibitors on ATIII, based on factor II. In these patients, chromogenic assays can be used, with accurate results. The same is true for dabigatran (a factor IIa inhibitor); A factor X-based chromogenic assay can be used. However, these specialized tests are expensive and not widely available.

Testing for the JAK2 V617F mutation involves PCR methods that are not affected by the use of DOACs. The diagnosis of paroxysmal nocturnal hemoglobinuria is made through a series of clinical and laboratory studies that should not be affected by the use of DOACs.

Effects of DOACs on testing for antiphospholipid syndrome

The diagnosis of APS is made in the presence of clinical and laboratory criteria. Laboratory criteria include testing for AL and anticardiolipin, IgM or IgG, or anti-β 2 -glycoprotein IgM or IgG, tested 12 weeks apart. The detection of anticardiolipin and antiβ 2 -glycoprotein antibodies are largely unaffected by DOAC use.

AL testing in the era of DOACs has many difficulties.

Many patients who present with thrombosis are anticoagulated in the acute setting, before performing thrombophilia tests. To complicate this further, if a patient is AL positive, another test must be performed 12 weeks later to confirm the diagnosis, at which time the action of the DOACs could already be established. The British Haematology Society guideline states that 2 different methods can be used to investigate AL. One is the TVVRD and another, the activated partial thromboplastin time (APTT), as a second test.

In general, the study of AL by TVVRD involves performing an initial screening test, which, if positive, is followed by a confirmatory test. Both steps can be affected by the use of DOACs. Screening values ​​using TVVRD have been shown to be high with rivaroxaban, apixaban, edoxaban and dabigatran, and some effects are observed in confirmatory tests.

In TAPT, the conversion of prothrombin to thrombin and the onset of clot formation depends on activated factor Some PTTA testing reagents are insensitive to DOACs, but this would require working with the laboratory and knowing what tests are available, and what DOACs were being used when interpreting the results.

Possible solutions to the effect of DOACs on thrombophilia testing

Since even at minimal DOAC levels thrombophilia testing appears to give accurate results, it has been suggested that such testing could be done at that point. However, data in this regard is lacking and there are still doubts in the interpretation of the results. Instead, the anticoagulant could be temporarily stopped. Only 5 half-lives of DOACs are needed to reduce its serum concentration to a level that does not affect coagulation tests for thrombophilia. In practice, this would mean stopping the DOAC for 2 to 3 days, which brings some problems, such as increased risk of thrombotic event in high-risk patients.

An alternative to stopping the anticoagulant drug would be to transition to LMWH for 24-48 hours. This would provide adequate anticoagulation with minimal interaction with the tests used. However, care should be taken when evaluating a patient with suspected AL and who is under the influence of LMWH.

Some reagents used to measure PTTA may be sensitive to LMWH at maximum levels; therefore, ideally, the test should be done with minimal levels of LMWH. It is convenient to be in communication with the local laboratory, but in practice it can be complicated, so it is not done often. It is also important to note that antithrombin levels may drop with LMWH use. Therefore, the TPTA may not be accurate.

An additional solution to testing would be to test for thrombophilias before starting a DOAC. This often has some problems. Anticoagulation is initiated immediately after diagnosis of thrombosis and delaying treatment can be harmful, while acute thrombosis can decrease plasma levels of proteins C and S and antithrombin.

In the case of LA testing, it is possible to use different laboratory assays that are not sensitive to DOACs. One such example is the Taipan snake venom time (TVST), which successfully detected LA in patients treated with rivaroxaban. However, not all ALs will be successfully detected and meet the criteria for diagnosis, and a second test would have to be used.

Only now have reagents been developed for use in PTTA tests, which are less sensitive to DOACs. It should be noted, however, that APS can be diagnosed in the presence of anticardiolipin IgG or IgM, or anti-β 2 -glycoprotein IgM or IgG antibodies, each taken 12 weeks apart. If the patient is already receiving a DOAC, LA testing will likely only be useful in diagnosing triple-positive APS, as it is high risk and will change management plans.

DOAC StopTM

The use of a new agent X9904-20, or DOAC-Stop TM (DOAC: direct acting oral anticoagulant or DOAC), has been proposed , which abrogates the effect of DOACs on thrombophilia testing. DOAC-Stop TM has been shown to adsorb rivaroxaban, apixaban, edoxaban and dabigatran from plasma. DOAC-Stop TM also achieves a statistically significant reduction in clotting time, in plasma samples dosed with rivaroxaban and apixaban, in the analysis of T, TAPT and factor VIII.

Therefore, DOAC-Stop TM can potentially be used to counteract the effects of factor Xa inhibitors in anticoagulated patients in thrombophilia testing. However, its use has drawbacks. While in most cases it allows accurate testing in patients taking apixaban, rivaroxaban and dabigatran, inappropriate results have been obtained in some with AL, observed in up to 3%.

At higher concentrations of rivaroxaban, apixaban and dabigatran, the effect of DOAC-Stop TM is limited and does not completely cancel the effect of the DOAC. One study also found that in different assays used in RPCA testing, the DOAC-StopTM had different effects on the results. An alternative to DOAC-Stop TM –DOAC-Remove TM (5D-82410A) – has given promising results to counteract the effect of DOACs in patients with and without thrombophilias.

Both agents could potentially be used to counteract the effects of factor Xa inhibitors for thrombophilia testing in anticoagulated patients, but there is currently insufficient evidence to support such use.

Can we give DOACs in thrombophilia?

The 2016 American College of Chest Physicians guidelines state that DOACs are preferable to VKAs for treating VTE, but do not provide guidance on the use of either class of medication for thrombophilia. Due to the lack of evidence to support the use of DOACs in patients with thrombophilia, doctors tend to use VKAs, to avoid unknown risks. Given the major advantages that DOACs have over them, there is an argument to evaluate the evidence for the safety of DOACs and their effectiveness in certain patients with thrombophilia.

There are inconsistent results regarding the safety of DOACs and their effectiveness in patients with thrombophilia. A meta-analysis has analyzed the safety of DOACs in various thrombophilias and compared the results with the use of VKAs and concluded that, especially for low-risk thrombophilia, DOACs were not inferior to VKAs, in terms of their efficacy and safety.

There is evidence of benefit from using ACOOD in patients with protein C, S and AT deficiencies but the evidence is largely anecdotal. Although it is thought that DOACs could be used in a selected subgroup of patients with low-risk paroxysmal nocturnal hematuria after thrombosis, due to lack of evidence, current clinical practice and recommendations, a VKA should be used instead.

JAK2 V617F mutations may be associated with myeloproliferative neoplasms, essential thrombocytosis, polycythemia vera, and myelofibrosis. It is generally accepted that managing the risk of thrombosis in these patients involves aggressive control of cardiovascular disease risk factors. Regarding whether DOACs can be used after thrombosis in this setting, VKA treatment is recommended.

Can we give DOACs in antiphospholipid syndrome?

In 2018, a large randomized controlled trial of warfarin vs. rivaroxaban in patients with high-risk APS demonstrated an increased risk of adverse events with rivaroxaban compared to warfarin, to the extent that the study had to be stopped before completion for safety reasons.

Subsequently, in 2020, the British Society of Haemtology updated the guidelines on APS. For patients with 3-antibody APS and VTE, treatment should be with warfarin with a contraindication to DOACs. If a patient is already treated with a DOAC and does not wish to switch, a DOAC is preferable to no treatment, but it is not optimal care. There are few treatment trials for patients with double-positive APS and VTE, but treatment with a VKA is recommended as the first choice.

Conclusion

There are no clear and conclusive guidelines regarding when thrombophilia testing should be done. Patients who have more risk factors for thrombophilia are a priority for thrombophilia testing, however, many guidelines suggest that tests should not be done if anticoagulant treatment is not interrupted.

Despite evidence showing that DOACs affect the tests used to evaluate inherited thrombophilias and APS, there are also no guidelines for how physicians should investigate thrombophilias in patients taking DOACs. Testing for patients taking a DOAC will give altered results.

Possible solutions to the effect of DOACs on thrombophilia testing are: temporarily discontinuing DOAC treatment, testing the patient at trough DOAC levels, switching the patient to LMWH, or using DOAC-Stop TM , although the literature is sparse. VKAs remain the treatment of choice in patients with triple-positive VTE and antibody-positive APS.

There is little evidence of the safety of DOACs in the secondary prevention of VTE associated with thrombophilias, but it is sufficient to recommend treatment. Until more evidence is available, the decision of whether a patient should be tested for thrombophilia, the timing of testing, and the therapeutic indication largely involves shared decision-making with the patient, carefully weighing the risks and benefits individually.